Methods and compositions for the production of sirnas

ABSTRACT

The technology described herein relates to siRNAs, e.g., methods and compositions relating to the production of siRNAs in bacterial cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.14/758,924, filed on Jul. 1, 2015, which is a 35 U.S.C. § 371 NationalPhase Entry Application of International Application No.PCT/US2014/010784 filed Jan. 9, 2014, which designates the U.S., andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 61/751,489 filed Jan. 11, 2013, the contents of whichare incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with federal funding under Grant Nos. AI087431awarded by the National Institutes of Health. The U.S. government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 9, 2014, isnamed 701039-075001-PCT_SL.txt and is 33,506 bytes in size.

TECHNICAL FIELD

The technology described herein relates to methods of producing siRNAsin vivo in bacterial cells.

BACKGROUND

RNA interference (RNAi) by double-stranded (ds) small interfering RNAs(siRNA) suppresses gene expression by inducing the degradation of mRNAsbearing complementary sequences (Fire, A. et al. Nature 1998391:806-811; Hamilton, A. J. & Baulcombe, D. C. Science 1999286:950-952). Transfection of synthetic siRNAs into eukaryotic cells tosilence genes has become an indispensable tool to investigate genefunction, and siRNA-based therapy is being developed to knockdown genesimplicated in disease (Elbashir, S. M. et al. Nature 2001, 411:494-8;Caplen, N. J., et al. Proc Natl Acad Sci USA 2001 98:97427; Rettig, G.R. & Behlke, M. A. Mol Ther 2012 20:483-512). More efficient ways toproduce siRNAs are desired.

SUMMARY

The technology described herein is directed to methods and compositionsrelating to the production of active siRNAs generated in vivo, e.g. inbacterial cells.

In one aspect, described herein is a bacterial cell comprising asiRNA-binding polypeptide and a dsRNA comprising a nucleic acid sequencesubstantially complementary to a target RNA. In some embodiments, thesiRNA-binding polypeptide comprises a purification tag. In someembodiments, the siRNA-binding polypeptide is encoded by a nucleic acid.In some embodiments, the siRNA-binding polypeptide is selected from thegroup consisting of: p19 polypeptide; tombusvirus p19 polypeptide; B2polypeptide; HC-Pro polypeptide; p38 polypeptide; p122 polypeptide; p130polypeptide; p21 polypeptide; p1b polypeptide; and NS3 polypeptide. Insome embodiments, the dsRNA is greater than 21 nucleotides in length. Insome embodiments, the dsRNA is a hairpin RNA. In some embodiments, thebacterial cell expresses an RNase III polypeptide. In some embodiments,the bacterial cell expresses an RNase III polypeptide encoded by anexogenous nucleic acid sequence. In some embodiments, the bacterial cellis an Escherichia coli cell. In some embodiments, at least one of thesiRNA-binding polypeptide and the dsRNA are constitutively expressed. Insome embodiments, at least one of the siRNA-binding polypeptide and thedsRNA are inducibly expressed. In some embodiments, the DNA encoding atleast one of the siRNA-binding polypeptide or the dsRNA is part of aplasmid.

In one aspect, described herein is a method of producing one or moresiRNA species which can inhibit the expression of a target RNA, themethod comprising: culturing a bacterial cell as described herein underconditions suitable for the production of siRNAs. In some embodiments,the method further comprises a second step of isolating thesiRNA-binding polypeptide and eluting the siRNAs bound to thesiRNA-binding polypeptide. In some embodiments, the method furthercomprises purifying the siRNAs eluted from the siRNA-binding polypeptideby chromatography e.g. anion exchange HPLC. In some embodiments, themethod further comprises contacting the cell with one or more modifiednucleotides before or during the culturing step.

In one aspect, described herein is a pharmaceutical compositioncomprising a siRNA produced according to the methods described herein.In some embodiments, the composition further comprises a population ofsiRNA species.

In one aspect, described herein is a pharmaceutical compositioncomprising a siRNA isolated from a bacterial cell as described herein.In some embodiments, the composition further comprises a population ofsiRNA species.

In one aspect, described herein is the use of a siRNA produced accordingto the methods described herein in the production of a medicament.

In one aspect, described herein is the use of a siRNA isolated from abacterial cell of as described herein in the production of a medicament.

In one aspect, described herein is a vector comprising; a nucleic acidencoding a siRNA-binding polypeptide; and a dsRNA cloning site. In someembodiments, the dsRNA cloning site comprises at least one restrictionenzyme site and can accept the insertion of at least one nucleic acidsequence such that a dsRNA is encoded and can be expressed. In oneaspect, described herein is a vector comprising: a nucleic acid encodinga siRNA-binding polypeptide; and a dsRNA comprising a nucleic acidsequence substantially complementary to a target RNA. In someembodiments, the siRNA-binding polypeptide is selected from the groupconsisting of: p19 polypeptide; tombusvirus p19 polypeptide; B2polypeptide; HC-Pro polypeptide; p38 polypeptide; p122 polypeptide; p130polypeptide; p21 polypeptide; p1b polypeptide; and NS3 polypeptide. Insome embodiments, the vector is a plasmid. In some embodiments, theplasmid further comprises a bacterial origin of replication.

In one aspect, described herein is a library of siRNA species, thelibrary comprising: a plurality of clonal bacterial cell populations;wherein each clonal population is comprises bacterial cells as describedherein. In one aspect, described herein is a library of siRNA species,the library comprising: a plurality of populations of siRNAs; whereineach population of siRNAs is obtained according to the methods describedherein. In some embodiments, each population of siRNAs binds to a singletarget RNA.

In one aspect, described herein is a kit comprising a bacterial cell asdescribed herein. In one aspect, described herein is a kit for theproduction of one or more species of siRNA, the kit comprising: abacterial cell comprising an siRNA-binding polypeptide; and at least onevector comprising a dsRNA cloning site. In one aspect, described hereinis a kit for the production of one or more species of siRNA, the kitcomprising: a bacterial cell comprising an siRNA-binding polypeptide;and at least one vector comprising a dsRNA comprising a nucleic acidsequence substantially complementary to a target RNA. In one aspect,described herein is a kit comprising a vector as described herein. Inone aspect, described herein is a kit for the production of one or morespecies of siRNA, the kit comprising two vectors; wherein the firstvector comprises a nucleic acid encoding a siRNA-binding polypeptide;and wherein the second vector comprises a dsRNA cloning site. In oneaspect, described herein is a kit for the production of one or morespecies of siRNA, the kit comprising two plasmids; wherein the firstvector comprises a nucleic acid encoding a siRNA-binding polypeptide;and wherein the second vector comprises a dsRNA comprising a nucleicacid sequence substantially complementary to a target RNA. In someembodiments, at least one vector is a plasmid. In some embodiments, theplasmid further comprises a bacterial origin of replication. In someembodiments, the kit further comprises a bacterial cell. In one aspect,described herein is a kit for the production of one or more species ofsiRNA, the kit comprising; a bacterial cell comprising a nucleic acidencoding a siRNA-binding polypeptide; and a vector comprising a dsRNAcloning site. In one aspect, described herein is a kit for theproduction of one or more species of siRNA, the kit comprising; abacterial cell comprising a nucleic acid encoding a siRNA-bindingpolypeptide; and a vector comprising a dsRNA comprising a nucleic acidsequence substantially complementary to a target RNA. In someembodiments, the siRNA-binding polypeptide comprises a purification tag.In some embodiments, the siRNA-binding polypeptide is encoded by anucleic acid. In some embodiments, the bacterial cell expresses an RNaseIII polypeptide. In some embodiments, the cell is an Escherichia colicell. In some embodiments, at least one of the siRNA-binding polypeptideand the dsRNA are operably linked to a constitutive promoter. In someembodiments, at least one of the siRNA-binding polypeptide and the dsRNAare operably linked to an inducible promoter. In some embodiments, theDNA encoding at least one of the siRNA-binding polypeptide or the dsRNAis part of a plasmid. In one aspect, described herein is a kitcomprising the library as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E demonstrate that ectopic p19 expression captures small RNAsin E. coli. FIG. 1A depicts an image of a gel from experiments in whichp19-coupled magnetic beads were used to isolate small dsRNAs from totalRNA isolated from mammalian ACH2 cells, or from E. coli cells, or fromE. coli cells containing a pcDNA3.1-P19 expression plasmid. CapturedRNAs were 5′ ³²P-labeled, separated on a native polyacrylamide gel anddetected by autoradiography. FIG. 1B depicts images of gels fromexperiments in which expression of FLAG-tagged p19, but not TREX1 orempty plasmid (V, vector), from pcDNA3.1+ in E. coli led to accumulationof ˜21 nt RNAs. Total RNAs purified from E. coli containing an emptyvector, or pcDNA3.1+ expressing FLAG-tagged p19 or TREX1 were separatedon a denaturing polyacrylamide gel and stained with SYBR Gold. FLAGimmunoblot verified protein expression. FIG. 1C depicts an image of agel from experiments in which total RNAs purified from E. colicontaining an empty vector, or pcDNA3.1+ expressing His-tagged p19 orHis-tagged p19 mutant proteins defective in RNA binding (Mut1¹⁴: W39G,W42G and Mut2¹⁶: K71A, R72G) were separated on a denaturingpolyacrylamide gel and stained with SYBR Gold. His immunoblot verifiedprotein expression. FIG. 1D depicts images of gels from experiments inwhich p19-associated RNAs were isolated with p19-coupled magnetic beadsfrom total RNA extracted from WT E. coli (DH5a or MG1655 Δlac) or RNaseIII mutant strains (in MG1655 Δlac background) expressing the His-taggedp19 protein. p19-captured RNAs were separated on native or denaturinggels and stained with SYBR Gold. p19 expression was verified by Hisimmunoblot. The asterisk (*) indicates equal loading of a backgroundband. FIG. 1E depicts an image of a gel from experiments in whichp19-associated RNAs were isolated with p19-coupled magnetic beads fromtotal RNA extracted from p19 expressing E. coli WT BL21(DE3) cells orrnc14 mutant HT115(DE3) cells co-transfected with p19 and pcDNA3.1vector that was empty or encoded Flag-tagged E. coli RNase III.p19-captured RNAs were separated on a native polyacrylamide gel andstained with SYBR Gold. p19 and RNase III expression was verified byimmunoblots. M, markers. Arrows indicate the ˜21 nt small RNA band. Dataare representative of at least 2 independent experiments.

FIGS. 2A-2F demonstrate pro-siRNAs knockdown EGFP expression. FIG. 2Adepicts a schematic of pGEX-4T-1-p19-T7 plasmid and the method toproduce pro-siRNAs from E. coli. FIG. 2B depict an image of a gel fromexperiments in which anion exchange HPLC fractions of SDS-eluted RNAs(isolated from E. coli transformed to express pro-siRNAs) were separatedon a native polyacrylamide gel and stained with SYBR Gold. FIG. 2Cdepicts images of gels from experiments in which nuclease sensitivityassay confirms pro-siRNAs are dsRNAs. Synthetic siRNAs and HPLC purifiedpro-siRNAs were incubated with nucleases and separated on a nativepolyacrylamide gel stained with SYBR Gold. FIG. 2D depicts images ofgels from experiments in which anti-Ago mAb 2A8 or mouse total IgG wasused to immunoprecipitate RNAs in negative control (NC) siRNA or EGFPFLpro-siRNA-transfected HeLa-d1EGFP cells. Immunoprecipitated RNAs wereanalyzed by Northern blot using a probe complementary to the full lengthEGFP coding sequence (top) or 5′ ³²P end-labeling (middle). Bottomimmunoblot is probed for Ago protein. FIG. 2E depicts a graph of theresults of qRT-PCR of EGFP expression and EGFP mean fluorescenceintensity by flow cytometry in HeLa-d1EGFP cells transfected with either4 nM of siRNA or pro-siRNAs. Data are normalized to cells treated withnegative control (NC) siRNA and are mean±SD of 2 (qRT-PCR) and 3 (EGFPfluorescence) independent experiments. mRNA level is relative to GADPH.FIG. 2F depicts images og gels from experiments in which HPLC-purifiedpro-siRNAs were separated on native or denaturing polyacrylamide gelsstained with SYBR Gold.

FIGS. 3A-3D demonstrate that pro-siRNA-mediated knockdown of endogenousand viral gene expression in human cells. FIG. 3A depicts graphs andimages of gels from experiments in which qRT-PCR and immunoblot assaysof genes targeted for knockdown with the indicated siRNAs or pro-siRNAs,which were transfected (4 nM) into HeLa-d1EGFP (top) or HCT116 (bottom)cells. FIG. 3B depicts graphs of cell counts after transfection withPLK1 siRNA or pro-siRNA (4 nM) or negative control (NC) siRNA or EGFPpro-siRNA as nontargeting controls, respectively. FIG. 3C depicts aschematic and graphs of the results of experiments demonstratinginhibition of HIV-1 spreading by transfection of vif siRNAs andpro-siRNAs (4 nM). vif siRNAs were either individually transfected (vifsiRNA-1 and vif siRNA-2) or co-transfected with 2 nM each of vif siRNA-1and siRNA-2 (vif siRNA1+2). (left) vif mRNA knockdown in HeLa-CD4 cells;(right) infectivity of culture supernatants from transfected HeLa-CD4cells by TZM-bl assay. FIG. 3D depicts graphs of the suppression ofmultiple HIV-1 strains by gag pro-siRNAs (transfected at 20 nM).Sequence of gagB siRNA (from IIIB) and its corresponding sequences inUG29 and IN22 strains were shown. (left) bar graph is gag mRNA knockdownin HeLa-CD4 cells (for IIIB), U87.CD4.CXCR4 cells (for UG29) andU87.CD4.CCR5 cells (for IN22); (right) infectivity of culturesupernatants by TZM-bl assay. Data are mean±SD of 3 (FIGS. 3A-3C) and 2(FIG. 3D) independent experiments. mRNA expression and TZM-bl luciferasedata are normalized to cells transfected with NC siRNA. FIG. 3Ddiscloses SEQ ID NOS 123-125, respectively, in order of appearance.

FIGS. 4A-4F demonstrating pro-siRNA sequences and tests of off-targeteffects. FIG. 4A depicts a graph of length distribution of EGFPFL,EGFP100 and LMNA pro-siRNAs assessed by deep sequencing. FIG. 4B depictsa graph of the percentage of sequence content of all aligned deepsequencing reads. FIG. 4C depicts graphs of the distribution of aligneddeep sequencing reads of EGFPFL, EGFP100 and LMNA pro-siRNAs. FIG. 4Ddepicts volcano plots of expression changes versus p value of allannotated transcripts detected by RNA deep sequencing in HeLa-d1EGFPcells transfected with EGFP siRNAs or pro-siRNAs relative to expressionin cells transfected with a negative control (NC) siRNA. Arrows indicateEGFP and the number is its fold change. Cut-off of significance isq_value<0.05 (default in Cufflinks). FIG. 4E depicts volcano plots ofexpression changes (1.2 fold less or more) versus p value detected bymicroarray in HeLa-d1EGFP cells transfected with LMNA siRNAs orpro-siRNAs relative to expression in cells transfected with a NC siRNA.Arrows indicate LMNA and the number is its fold change. Cut-off ofsignificance is p<0.05 (by paired T-test). FIG. 4F depicts a graph ofthe percentage of significantly changed transcripts in FIGS. 4D-4E.

FIGS. 5A-5B demonstrate that ectopic expression of p19 stabilizes ˜21 ntsmall RNA species in Listeria monocytogenes. FIG. 5A depicts an image ofgel demonstrating that approximately 21 nt small RNAs co-purify withp19. L. monocytogenes was transformed with an empty vector (pLIV-1) orwith pLIV-1 encoding inducible N-terminal His-tagged p19(pLIV-1-p19-His). Duplicate cultures were grown in the presence of IPTGto induce protein expression. Samples were then lysed and incubated withNi resin to purify the His-p19 protein and any associated RNAs.p19-bound RNAs were separated on a denaturing polyacrylamide gel stainedwith SYBR gold. M, RNA markers. Arrow indicates ˜21 nt small RNAs. FIG.5B depicts an image of an immunoblot with His antibody to confirmIPTG-dependent induction of His-p19.

FIGS. 6A-6D demonstrate that SDS efficiently elutes GST-p19-His-boundsmall RNAs but not GST-p19-His protein. FIG. 6A depicts an image of gelfrom an experiment in which GST-p19-His protein, induced in E. coli withIPTG and purified by imidazole elution from Ni resin, was assayed bySDS-PAGE and Coomassie blue staining FIG. 6B depicts an image of a geldemonstrating that imidazole, but not SDS (0.5%), elutes GST-p19-Hisprotein from Ni beads. Coomassie blue staining of proteins eluted fromNi resin with imidazole (lane 1) or SDS (lane 2). Lane 3 shows proteinsbound to the Ni resin before any elution, lane 4 is a sample of the SDSeluate and lane 5 shows proteins remaining on the beads after SDSelution (lane 5). M, protein markers. FIG. 6C depicts an image of a geldemonstrating that SDS elution efficiently elutes GST-p19-His-boundsmall RNAs. Ni resin was boiled before or after incubation with 0.5% SDSand bound RNAs were analyzed on a denaturing polyacrylamide gel stainedwith SYBR Gold. Arrow indicates ˜21 nt small RNAs, which were removed bySDS treatment. FIG. 6D depicts a schematic summary of the effect ofimidazole or SDS elution of material captured by Ni resin from E. coliexpressing GST-p19-His protein.

FIGS. 7A-7B demonstrate the dose response comparison of gene silencingby EGFP siRNAs and pro-siRNAs and test of antisense EGFP construct. FIG.7A depicts a graph of EGFP fluorescence in HeLa-d1EGFP cells transfectedwith either siRNAs or pro-siRNAs at the indicated concentrations. Dataare a representative dose-response experiment. FIG. 7B depictsschematics and a graph. Schemes of empty, EGFP antisense and hairpinplasmids used to produce pro-siRNAs. Bar graph is the percentage of EGFPexpressing HeLa-d1EGFP cells after transfection of NC siRNA andpro-siRNAs (at 0.5 nM). Data are mean±SD of 2 independent transfections.

FIGS. 8A-8B demonstrate that pro-siRNA knockdown of gene expression isindependent of Dicer. FIG. 8A depicts a graph of Negative control (NC)siRNA, EGFP siRNA or EGFPFL pro-siRNA co-transfected with pEGFP-N1plasmid into HCT116 cells that contained a Dicer exon 5 deletionmutation (HCT116 Dicer^(−/−))¹⁹ EGFP knockdown by siRNAs or pro-siRNAs,as measured by flow cytometry, occurred in Dicer-deficient cells. Dataare representative of 3 independent experiments. FIG. 8B depicts animage of a gel from experiments in which double stranded RNAs, siRNAsand pro-siRNAs were incubated or not with recombinant Dicer protein for18 hrs at 37° C. Resulting products were separated on a 20%polyacrylamide gel and stained with SYBR Gold.

FIG. 9 depicts graphs demonstrating dose response comparison of genesilencing by pro-siRNAs and commercial siRNAs. Total RNAs were extractedfrom HeLa-d1EGFP cells 24 hrs after transfection. mRNA levels werenormalized to negative control siRNA transfected cells. Two independentexperiments were shown. Damachon siRNAs: siRNA-D1-D4. siRNA of publishedsequence: siRNA-G.

FIGS. 10A-10B demonstrate that pro-siRNAs induce little expression ofproinflammatory cytokines in primary monocyte-derived human macrophages.FIG. 10A depicts a graph of the results of a qRT-PCR assay of theindicated proinflammatory cytokine gene mRNAs, 4 hr after treatment withthe indicated concentrations of LPS, synthetic siRNAs, HPLC-purifiedpro-siRNAs or SDS-eluate. mRNA levels were normalized to levels inuntreated cells. FIG. 10B depicts a graph of the results of a qRT-PCRassay of the indicated proinflammatory cytokine gene, LMNA and IFIT1mRNAs, 24 hrs after transfection with indicated siRNA and pro-siRNA (at20 nM). PolyI:C was used as positive control and mRNA levels werenormalized to levels in mock transfected cells.

FIG. 11 depicts length profile and distribution of deep sequencing readsaligned to the pro-siRNA target sequences.

FIGS. 12A-12E demonstrate a test of strand bias and validation ofpro-siRNA ‘hot spots’ for EGFPFL pro-siRNA. FIG. 12A depicts a graph ofthe position of DNA oligonucleotides (26-27 nt) used for probing EGFPFLsmall RNAs compared to position of aligned sequencing reads. The linearscale emphasizes sequencing hot spots. F, forward probe: R, reverseprobe. FIG. 12B depicts images of gels from experiments in whichpurified pro-siRNAs were denatured and incubated with the indicated DNAprobes, and then the reaction mixture was analysed for the formation ofDNA:RNA hybrids by native polyacrylamide gel electrophoresis andautoradiography. (top) short exposure; (middle) long exposure; (bottom)DNA oligonucleotides only, exposed for 1 hr, to show comparablelabelling. Arrows indicate the DNA:RNA hybrids. FIG. 12C depicts a graphof band intensities from FIG. 12B which were quantified usingMulti-gauge software (Fujifilm); FIG. 12D depicts a graph of the ratioof sense to antisense signal for each pair of probes calculated bydividing the DNA:RNA hybrid band intensities detected with the “R”oligonucleotide by that detected with the “F” oligonucleotide. FIG. 12Edepicts a graph of normalized levels (to Si1) of hybridization signals(from FIG. 12C) and numbers of deep sequencing reads (from Table 2) ofthe three hot spots.

FIGS. 13A-13E demonstrate the similarity of EGFPFL pro-siRNA sequencecontents and hot spot patterns obtained in two independent pro-siRNApreparations. Graphs are depicted, comparing gene knockdown of EGFPmeasured by flow cytometry (FIG. 13A) sequence content (FIG. 13B),length profile (FIG. 13C) and distribution (FIG. 13D) of deep sequencingreads of two independent EGFPFL pro-siRNAs (EGFPFL-1 and EGFPFL-2). FIG.13E depicts a graph comparing deep sequencing reads profiles ofpro-siRNAs made from top (1-360 nt, Hotspot-1) or bottom half of EGFP(361-720 nt, Hotspot-2) with pro-siRNAs made from full length EGFP(1-720 nt, EGFPFL-1). In (13D-13E) dashed lines and * highlighted sharedhotspots. NC, negative control siRNA

FIGS. 14A-14D demonstrate the off-target effect of siRNAs andpro-siRNAs. FIG. 14D depicts Venn diagrams for significantly changedgenes in HeLa-d1EGFP cells transfected with EGFP siRNAs or pro-siRNAs.FIG. 14B depicts volcano plots of expression changes versus p value ofall annotated lincRNA by RNA deep sequencing in HeLa-d1EGFP cellstransfected with EGFP siRNAs or pro-siRNAs relative to expression incells transfected with a negative control (NC) siRNA. Cut-off ofsignificance is q_value<0.05 (default in Cufflinks). FIG. 14C depictsgraphs of the number of significantly changed lincRNAs. FIG. 14D depictsVenn diagrams for significantly changed genes in HeLa-d1EGFP cellstransfected with LMAN siRNAs or pro-siRNAs.

FIGS. 15A-15C demonstrate a two-plasmid alternate method for generatingpro-siRNAs in E. coli. FIG. 15A depicts a schematic of method to producepro-siRNAs in E. coli using a two plasmid approach, where one plasmiddirects p19 expression and the other expression of dsRNA correspondingto the target sequence. FIG. 15B depicts and image of a gel fromexperiments in which SDS eluate of pro-siRNAs targeting EGFP, producedusing this two-plasmid approach from bacteria transformed with eitherpRSF-GST-p19-His or pCDF-GST-p19-His (encoding GST-p19-His fusionprotein) in combination with L4440-EGFP plasmid (encoding T7-drivensense and antisense EGFP transcripts), were separated on a nativepolyacrylamide gel stained with SYBR Gold. FIG. 15C depicts graphs ofEGFP fluorescence measured by flow cytometry in HeLa-d1EGFP cellstransfected with indicated siRNA or pro-siRNA (˜10 nM). Data arerepresentative of 3 independent experiments.

FIG. 16 demonstrates an exemplary method to improve yield of pro-siRNAs.pGEX-4T-1-p19-T7 plasmid containing EGFP hairpin (used to make EGFPFLpro-siRNA) was co-transfected with p19 overexpressing plasmids (pCDF-p19or pRSF-p19) or E. coli RNase III overexpressing plasmid (pCDF-RNaseIII). The two-plasmid system (FIGS. 15A-15C) of co-transfectingL4440-EGFP with pCDF-p19 or pRSF-p19 was also tested. All E. coli cellswere cultured under the same conditions. pro-siRNAs were produced as inFIG. 2A and equal proportions of SDS eluate were separated on a nativepolyacrylamide gel and stained with SYBR Gold. ˜21 nt small RNA band wasquantified using Gel Logic software and signals were normalized to theband in the first sample lane. Total RNA samples extracted from E. colicells of each condition, treated with or without RNase A, were separatedon a 0.8% agarose gel containing EtBr. Immunoblots were performed toconfirm expressing of p19 and RNase III. M, molecular weight marker.

DETAILED DESCRIPTION

Embodiments of the invention described herein are directed to methodsand compositions relating to the production of siRNAs in vivo, e.g. inbacterial cells (siRNAs produced according to the methods andcompositions described herein are also referred to herein as“pro-siRNAs”). The technology described herein is derived from theinventors' discovery that prokaryotic cells have the ability to generatesiRNAs (e.g. pro-siRNAs). As prokaryotic cells are not known to expresscomponents of the canonical RNAi machinery (e.g. Dicer), it waspreviously believed that prokaryotic cells were incapable of producingsiRNAs.

As described in the Examples herein, when the inventors isolated p19polypeptide which was expressed in a prokaryotic cell, it was found thatthe p19 polypeptide was bound to siRNAs present in the prokaryotic cell(pro-siRNAs). In the absence of the exogenous p19 polypeptide, thesesiRNAs are undetectable. These results indicated, in contrast toexisting consensus in the field, that prokaryotic cells are capable ofgenerating siRNAs, even in the absence of the canonical siRNA machinery,e.g. Dicer. The results further indicate that the endogenous siRNAs haveexceptionally short half-lives which prevent their detection and/orisolation. When the inventors expressed both a p19 polypeptide and adsRNA having sequence complementary to a target RNA in the prokaryoticcell, siRNAs specific for the target RNA were generated by theprokaryotic cell. The activity of these siRNAs in silencing the targetRNA expressed by a eukaryotic cell is demonstrated herein.

Embodiments described herein use endogenous biological processes togenerate siRNAs from dsRNA, not requiring the use of algorithms whichattempt to predict efficacious siRNA sequences. Embodiments describedherein also relate to populations of multiple siRNA species, wherein thepopulation as a whole is specific for a target RNA. Such populations ofsiRNA species can have reduced off-target effects and greater efficacythan single RNA species.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, the terms “reduced”, “reduction”, “decrease”, or “inhibit”can mean a decrease by at least 10% as compared to a reference level,for example a decrease by at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or more orany decrease of at least 10% as compared to a reference level. In someembodiments, the terms can represent a 100% decrease, i.e. anon-detectable level as compared to a reference level. In the context ofa marker or symptom is meant a statistically significant decrease insuch level. The decrease can be, for example, at least 10%, at least20%, at least 30%, at least 40% or more, and is preferably down to alevel accepted as within the range of normal for an individual withoutsuch disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments, the subject is a mammal, e.g., a primate, e.g., ahuman. The terms, “individual,” “patient” and “subject” are usedinterchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. A subject can be male or female.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, the term “proteins” and “polypeptides” are usedinterchangeably herein to designate a series of amino acid residuesconnected to the other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one strand nucleic acid of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the template nucleic acid is DNA. In another aspect, thetemplate is RNA. Suitable nucleic acid molecules are DNA, includinggenomic DNA or cDNA. Other suitable nucleic acid molecules are RNA,including mRNA.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, transcript processing, translation and protein folding,modification and processing. “Expression products” include RNAtranscribed from a gene, and polypeptides obtained by translation ofmRNA transcribed from a gene. The term “gene” means the nucleic acidsequence which is transcribed (DNA) to RNA in vitro or in vivo whenoperably linked to appropriate regulatory sequences. A gene may or maynot include regions preceding and following the coding region, e.g. 5′untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer”sequences, as well as intervening sequences (introns) between individualcoding segments (exons).

The term “operatively linked” includes having an appropriate startsignal (e.g., ATG) in front of the polynucleotide sequence to beexpressed, and maintaining the correct reading frame to permitexpression of the polynucleotide sequence under the control of theexpression control sequence, and, optionally, production of the desiredpolypeptide encoded by the polynucleotide sequence. In some examples,transcription of a nucleic acid modulatory compound is under the controlof a promoter sequence (or other transcriptional regulatory sequence)which controls the expression of the nucleic acid in a cell-type inwhich expression is intended. It will also be understood that themodulatory nucleic acid can be under the control of transcriptionalregulatory sequences which are the same or which are different fromthose sequences which control transcription of the naturally-occurringform of a protein.

The term “isolated” or “partially purified” as used herein refers, inthe case of a nucleic acid or polypeptide, to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) that is present with the nucleic acid orpolypeptide as found in its natural source and/or that would be presentwith the nucleic acid or polypeptide when expressed by a cell, orsecreted in the case of secreted polypeptides. A chemically synthesizednucleic acid or polypeptide or one synthesized using in vitrotranscription/translation is considered “isolated.”

As used herein, the term “exogenous” refers to a substance (e.g. anucleic acid or polypeptide) present in a cell other than its nativesource. The term exogenous can refer to a nucleic acid or a protein(that has been introduced by a process involving the hand of man into abiological system such as a cell or organism in which it is not normallyfound or in which it is found in undetectable amounts. A substance canbe considered exogenous if it is introduced into a cell or an ancestorof the cell that inherits the substance. In contrast, the term“endogenous” refers to a substance that is native to the biologicalsystem or cell.

As used herein, the term “complementary” or “complementary base pair”refers to A:T and G:C in DNA and A:U in RNA. Most DNA consists ofsequences of nucleotide only four nitrogenous bases: base or baseadenine (A), thymine (T), guanine (G), and cytosine (C). Together thesebases form the genetic alphabet, and long ordered sequences of themcontain, in coded form, much of the information present in genes. MostRNA also consists of sequences of only four bases. However, in RNA,thymine is replaced by uridine (U).

As used herein, “substantially complementary” refers to a firstnucleotide sequence having at least 90% complementarity over the entirelength of the sequence with a second nucleotide sequence, e.g. 90%complementary, 95% complementary, 98% complementary, 99% complementary,or 100% complementary. Two nucleotide sequences can be substantiallycomplementary even if less than 100% of the bases are complementary,e.g. the sequences can be mismatched at certain bases.

As used herein, the terms “gene silencing”, “silencing”, or “RNAi” referto a phenomenon where an agent for causing RNAi, such as double-strandedRNA (dsRNA) causes the specific degradation of homologous RNA, thussuppressing the expression of gene products (see Coburn, G. and Cullen,B. (2002) J. of Virology 76:9225). This process has been described inplants, invertebrates, and mammalian cells. An RNAi agent can besubstantially homologous to the target RNA gene or genomic sequence, ora fragment thereof. As used in this context, the term “homologous” isdefined as being substantially identical, sufficiently complementary, orsimilar to the target RNA, or a fragment thereof, to effect RNAinterference of the target RNA. In addition to native RNA molecules,RNAs suitable for inhibiting or interfering with the expression of atarget RNA include RNA derivatives and analogs. RNAi can be caused byany type of interfering RNA, including but are not limited to, siRNA,shRNA, endogenous microRNA and artificial microRNA. In some embodiments,the RNAi molecule is a small interfering RNA (siRNA). An RNAi agent cancause a decrease in the level of a target RNA in a cell by at leastabout 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 95%, about 99% or more ofthe target RNA level found in the cell without the presence of the genesilencing agent. In one preferred embodiment, the target RNA levels aredecreased by at least about 70%, about 80%, about 90%, about 95%, about99% or more.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorder.Treatment is generally “effective” if one or more symptoms or clinicalmarkers are reduced. Alternatively, treatment is “effective” if theprogression of a disease is reduced or halted. That is, “treatment”includes not just the improvement of symptoms or markers, but also acessation of, or at least slowing of, progress or worsening of symptomscompared to what would be expected in the absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, remission (whether partial or total), and/or decreasedmortality, whether detectable or undetectable. The term “treatment” of adisease also includes providing relief from the symptoms or side-effectsof the disease (including palliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in “The Merck Manual of Diagnosis and Therapy”, 19th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology,published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9).Definitions of common terms in molecular biology can also be found inBenjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009(ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols inProtein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention can be performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); Current Protocols in Cell Biology (CPCB) (Juan S.Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture ofAnimal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher:Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods inCell Biology, Vol. 57, Jennie P. Mather and David Barnes editors,Academic Press, 1st edition, 1998) which are all incorporated byreference herein in their entireties.

Other terms are defined herein within the description of the variousaspects of the invention.

Embodiments of the technology described herein include methods andcompositions relating to a bacterial cell comprising a siRNA-bindingpolypeptide and a dsRNA; wherein the dsRNA comprises a nucleic acidsequence substantially complementary to at least one target RNA.siRNA-generating enzymes (e.g. RNAses) present in the bacterial cell(e.g. either endogenous or exogenous) can generate siRNA molecules fromthe dsRNA, which can then be bound by the siRNA-binding polypeptide. Thebinding of the siRNA-binding polypeptide can enable purification of thesiRNA molecules from the other constituents of the bacterial cell andprevent further degradation of the siRNA to non-siRNA substituents, e.g.dsRNAs of less than 15 nucleotides in length or individualribonucleotides.

As used herein, “double stranded RNA” or “dsRNA” refers to RNA moleculesthat comprise two substantially complementary strands. Double-strandedmolecules include those comprising a single RNA molecule that doublesback on itself to form a two-stranded structure, e.g. a stem-loopmolecule or a hairpin molecule. In some embodiments, a dsRNA cancomprise nucleic acid sequences which are not substantiallycomplementary to other sequences of the dsRNA (i.e. a single-strandedportion of the dsRNA), for example, the loop part of a stem-loopstructure. The portion of the dsRNA which comprises a nucleic acidsequence substantially complementary to a target RNA should comprise, atleast in part, the double-stranded portion of a dsRNA. In someembodiments, the double-stranded portion of a dsRNA comprising a nucleicacid sequence substantially complementary to at least one target RNA canbe 21 nucleotides in length or greater, e.g. 21 nucleotides or greater,22 nucleotides or greater, 23 nucleotides or greater, 24 nucleotides orgreater, 25 nucleotides or greater, 50 nucleotides or greater, 100nucleotides or greater, 200 nucleotides or greater, 500 nucleotides orgreater, 1000 nucleotides or greater in length.

As used herein, the term “target RNA” refers to a RNA present in a cell(i.e. the “target cell”). The target RNA comprises a target sequence towhich one strand of a siRNA according to the methods and compositionsdescribed herein binds, thereby causing RNAi silencing of the targetRNA. The target cell can be the bacterial cell comprising asiRNA-binding polypeptide or another cell, either prokaryotic oreukaryotic. The target sequence can be an RNA that can be translated(i.e. it can encode a polypeptide, e.g. mRNA) or it can be an RNA thatis not translated (i.e. a non-coding RNA). In some embodiments, thetarget sequence can be any portion of an mRNA. In some embodiments, thetarget sequence can be a sequence endogenous to the cell. In someembodiments, the target sequence can be a sequence exogenous to thecell. In some embodiments, the target sequence can be sequence from anorganism that is pathogenic to the target cell, e.g. the target sequencecan be sequence from a viral, bacterial, fungal, and/or parasiticorigin. In some embodiments, the target sequence is a viral nucleotidesequence.

In some embodiments, a nucleic acid sequence substantially complementaryto a target RNA can comprise a nucleic acid sequence substantiallycomplementary to part or all of the sequence of the target RNA. In someembodiments, a dsRNA comprising a nucleic acid sequence substantiallycomplementary to a target RNA can comprise sequence complementary topart or all of a specific allele, variant, and/or mutation (e.g.,insertions, deletions, fusions, SNPs, etc.) of a target RNA. In someembodiments, the dsRNA comprising a nucleic acid sequence substantiallycomplementary to a target RNA can comprise nucleic acid sequence(s)substantially complementary to multiple target RNAs (e.g. target RNAsencoding separate genes or target RNAs encoding multiple variants of thesame gene). In some embodiments, a dsRNA comprising a nucleic acidsequence substantially complementary to a target RNA can comprise anucleic acid sequence substantially complementary to all or part of oneor more exons of a target mRNA. In some embodiments, a dsRNA comprisinga nucleic acid sequence substantially complementary to a target RNA cancomprise a nucleic acid sequence substantially complementary to a cDNA.In some embodiments, a dsRNA comprising a nucleic acid sequencesubstantially complementary to a target RNA can comprise a nucleic acidsequence (or its complement) obtained from the transcriptome and/orgenome of a cell.

In some embodiments, the dsRNA can comprise two separate complementarystrands, e.g. a sense and antisense strand.

In some embodiments, the dsRNA can be a hairpin RNA, i.e. an RNAcomprising two portions which are reverse complements, separated by asequence which will not self-anneal, thus forming a stem-loop or“hairpin” structure. In some embodiments, the double-stranded portion ofa hairpin RNA can be at least 19 nucleotides in length. In someembodiments, the double-stranded portion of a hairpin RNA can be atleast 25 nucleotides in length. In some embodiments, the double-strandedportion of a hairpin RNA can be 30 nucleotides in length or greater,e.g. at least 30 nucleotides, at least 50 nucleotides, at least 100nucleotides, at least 200 nucleotides, or at least 300 nucleotides. Insome embodiments, the dsRNA can be a shRNA. As used herein “shRNA” or“small hairpin RNA” (also called stem loop) is a type of dsRNA. In oneembodiment, these shRNAs are composed of a short, e.g. about 19 to about25 nucleotide, antisense strand, followed by a nucleotide loop of about5 to about 9 nucleotides, and the analogous sense strand. Alternatively,the sense strand can precede the nucleotide loop structure and theantisense strand can follow.

In some embodiments, increased length of the double-stranded portion ofa dsRNA can correlate with a decreased level of off-target effects, e.g.silencing of non-targeted genes. In some embodiments, one strand of thedouble-stranded portion of a dsRNA can be at least 100 nucleotides inlength. For example, one strand of the double-stranded portion of adsRNA can be at least 100 nucleotides in length, at least 200nucleotides in length, at least 300 nucleotides in length, at least 400nucleotides in length, at least 500 nucleotides in length, at least 700nucleotides in length, or at least 1000 nucleotides in length.

In some embodiments, the dsRNA can be exogenous to the cell. In someembodiments, the target sequence of the target RNA can be exogenous tothe cell. In some embodiments, the target RNA can be exogenous to thecell. In some embodiments, the nucleic acid sequence substantiallycomplementary to a target RNA can be exogenous to the cell.

In the methods and compositions described herein, siRNAs can begenerated from the dsRNA comprising a nucleic acid sequencesubstantially complementary to a target RNA. As used herein, the term“siRNA” refers to a nucleic acid that forms an RNA molecule comprisingtwo individual strands of RNA which are substantially complementary toeach other. Typically, the siRNA is at least about 15-40 nucleotides inlength (e.g., each complementary sequence of the double stranded siRNAis about 15-40 nucleotides in length, and the double stranded siRNA isabout 15-40 base pairs in length, preferably about 19-25 basenucleotides, e.g., 19, 20, 21, 22, 23, 24, or 25 nucleotides in length).In some embodiments, a siRNA can be blunt-ended. In some embodiments, asiRNA can comprise a 3′ and/or 5′ overhang on each strand having alength of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of theoverhang is independent between the two strands, i.e., the length of theoverhang on one strand is not dependent on the length of the overhang onthe second strand. The siRNA molecules can also comprise a 3′ hydroxylgroup. In some embodiments, the siRNA can comprise a 5′ phosphate group.A siRNA has the ability to reduce or inhibit expression of a gene ortarget RNA when the siRNA is present or expressed in the same cell asthe target gene, e.g. the target RNA. siRNA-dependentpost-transcriptional silencing of gene expression involves cutting thetarget RNA molecule at a site guided by the siRNA.

In some embodiments, a single siRNA species can be generated from adsRNA. In some embodiments, multiple siRNA species can be generated froma dsRNA. For example, two or more siRNA species can be generated from adsRNA, e.g. two or more siRNA species, three or more siRNA species, fiveor more siRNA species, or ten or more siRNA species. As used herein, theterm “a siRNA species” refers to one or more siRNA molecules which areidentical in sequence. In embodiments where multiple siRNA species aregenerated from a single dsRNA, the species can comprise sequencecomplementary to the same target RNA or to separate target RNAs. In someembodiments, a single dsRNA can comprise sequence complementary tomultiple target RNAs. In some embodiments, a single dsRNA can comprisemultiple sequences, each of which is complementary to a unique targetRNA, e.g. a multiplicity of siRNA species targeting (e.g. complementaryto) a multiplicity of target RNAs can be generated from a single dsRNA.

In the methods and compositions described herein, a dsRNA present withina bacterial cell can be cleaved to generate one or more siRNA species.The siRNA molecules can then be bound by a siRNA-binding polypeptidealso present within the bacterial cell. As used herein, the term“siRNA-binding polypeptide” refers to a polypeptide capable of bindingto siRNAs and increasing the half-life or detectable level of siRNAs ina prokaryotic cell. In some embodiments, the siRNA-binding polypeptidecan bind preferentially or specifically to siRNAs as compared to otherdsRNA species, e.g. the polypeptide can bind preferentially orspecifically to siRNAs as compared to dsRNAs greater than 25 or lessthan 15 nucleotides in size. In some embodiments, the siRNA-bindingpolypeptide can bind preferentially or specifically to siRNAs ascompared to other dsRNA species, e.g. dsRNAs greater than 25 or lessthan 15 nucleotides in size. In one embodiment, the siRNA-bindingpolypeptide does not bind to dsRNA having a double-stranded portionlonger than 25 nucleotides in length. In some embodiments, thesiRNA-binding polypeptide can bind preferentially or specifically tosiRNAs as compared to single-stranded RNA species.

In some embodiments, a siRNA-binding polypeptide can detectably bind toa siRNA. In some embodiments, a siRNA-binding polypeptide can be apolypeptide that when expressed in a bacterial cell, can causedetectable levels of siRNAs to be present in that cell when detectablelevels of siRNAs are not present in the wild-type bacterial cell. Insome embodiments, a siRNA-binding polypeptide can be a polypeptide thatincreases the half-life or detectable level of siRNAs in a prokaryoticcell by at least 5%, e.g. by at least 5%, by at least 10%, by at least20%, by at least 30%, by at least 50%, by at least 75%, by at least100%, by at least 200% or more.

In some embodiments, a siRNA-binding polypeptide can be a p19polypeptide. As used herein, the term “p19” refers to a viral proteinwhich binds specifically to dsRNAs and which suppresses RNAi-mediatedhost plant viral defenses. The sequences of p19 polypeptides from anumber of species are known, e.g. tombusvirus p19 (NCBI Gene ID:1493957; SEQ ID NO:1). In some embodiments, the p19 polypeptide can betombusvirus p19. Non-limiting examples of p19 homologues includeCarnation Italian ringspot virus P19; Tomato bushy stunt virus p19;Artichoke mottled crinkle virus p19; Lisianthus necrosis virus p19; Pearlatent virus p19; Cucumber Bulgarian virus p19; Cucumber necrosis virusp19; Pelargonium necrotic spot virus p19; Cymbidium ringspot virus p19;Lisianthus necrosis virus p19; Lettuce necrotic stunt virus p19; Maizenecrotic streak virus p19; Grapevine Algerian necrosis virus p19; andGrapevine Algerian latent virus p19. A p19 polypeptide can comprisemutants, variants, homologues, and functional fragments of wildtype p19polypeptides.

Further non-limiting examples of an siRNA-binding polypeptide caninclude the Flock house virus B2; HC-Pro; Tobacco etch virus HC-Pro;P38; P122; P130; Tobamovirus P122/P130; p21; Rice hoja blanca tenuivirus(RHBV) NS3; Cucumber vein yellowing virus P1b; HC-Pro of potyviruses;p21 of Beet yellows virus and Closterovirus; and variants, homologues,or functional fragments of the foregoing.

In some embodiments, an siRNA-binding polypeptide can be anenzymatically inactive member of the RISC complex, e.g. an enzymaticallyinactive variant or mutant of Argonaute or Dicer (see, e.g. Buker et al.Nat Struct Mol Bio 2007 14:200-7 and Liu et al. Molecular Cell 201246:1-11; which are incorporated by reference herein in theirentireties). In some embodiments, the siRNA-binding polypeptide is notan enzymatically active member of the RISC complex, e.g. an Argonaute orRISC polypeptide. As used herein, the term “RISC complex” refers to theproteins and single-stranded polynucleotides that interact to recognizetarget RNA molecules. Demonstrated components of RISC include theArgonaute proteins (e.g. Aubergine; Argonaute 2), R2D2, and Dicer (e.g.Der-2). In the case of an active. RISC complex loaded with asingle-stranded guide RNA derived from a siRNA, the RISC complex cancleave the target RNA molecule.

In some embodiments, a siRNA-binding polypeptide can be a polypeptidethat can bind to nucleic acids, e.g. protamine, or a variant, homologue,or functional fragment thereof (see, e.g. Rossi. Nature Biotechnology2005 23:682-4 and Reischl et al. Scientia Pharmaceutica 2010 78:686;which are incorporated by reference herein in their entirety). In someembodiments, a siRNA-binding polypeptide can be a polypeptide that canbe bind to dsRNAs, e.g. TARBP2 or a polypeptide comprising adouble-stranded RNA binding domain (see, e.g. US Patent Publication2009/0093026; which is incorporated by reference herein in its entirety)or a variant, homologue, or functional fragment thereof.

A functional fragment of a siRNA-binding polypeptide can be any portionof a siRNA-binding polypeptide which retains at least 50% of thewild-type level of siRNA binding activity, e.g. at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least98%, or more.

In some embodiments, a siRNA-binding polypeptide can comprise apurification tag. The term “purification tag” as used herein refers toany peptide sequence suitable for purification of a siRNA-bindingpolypeptide, and optionally, siRNAs bound by the siRNA-bindingpolypeptide. The purification tag specifically binds to (or is bound by)another moiety with affinity for the purification tag. Such moietieswhich specifically bind to a purification tag can be attached to amatrix or a resin, e.g. agarose beads. Moieties which specifically bindto purification tags can include antibodies, nickel or cobalt ions orresins, biotin, amylose, maltose, and cyclodextrin. Exemplarypurification tags can include histidine tags (such as a hexahistidinepeptide (SEQ ID NO: 122)), which will bind to metal ions such as nickelor cobalt ions. Therefore, in certain embodiments the purification tagcan comprise a peptide sequence which specifically binds metal ions.Other exemplary purification tags are the myc tag (EQKLISEEDL (SEQ IDNO:3)), the Strep tag (WSHPQFEK (SEQ ID NO:4)), the Flag tag (DYKDDDDK(SEQ ID NO:5)) and the V5 tag (GKPIPNPLLGLDST (SEQ ID NO:6)). The term“purification tag” also includes “epitope tags”, i.e. peptide sequenceswhich are specifically recognized by antibodies. Exemplary epitope tagscan include the FLAG tag, which is specifically recognized by amonoclonal anti-FLAG antibody. The peptide sequence recognized by theanti-FLAG antibody consists of the sequence DYKDDDDK (SEQ ID NO: 5) or asubstantially identical variant thereof. Therefore, in certainembodiments the purification tag can comprise a peptide sequence whichis specifically recognized by an antibody. The term “purification tag”also includes substantially identical variants of purification tags.“Substantially identical variant” as used herein refers to derivativesor fragments of purification tags which are modified compared to theoriginal purification tag (e.g. via amino acid substitutions, deletionsor insertions), but which retain the property of the purification tag ofspecifically binding to a moiety which specifically recognizes thepurification tag. In some embodiments, the siRNA-binding polypeptide canbe a p19 fusion protein as described in US Patent Publication2010/0209933; which is incorporated herein by reference in its entirety.

In some embodiments, the siRNA-binding polypeptide can be encoded by anucleic acid present in the bacterial cell, i.e. the polypeptide istranscribed and translated by the bacterial cell. In some embodiments,the siRNA-binding polypeptide can be introduced into the bacterial cellas a polypeptide. Uptake of polypeptides can be induced by any means inthe art. Non-limiting examples include the protocols described inShellman and Pettijohn. J Bacteriology 1991 173:3047-3059; which isincorporated by reference herein in its entirety.

In some embodiments, a nucleic acid encoding a siRNA-binding polypeptideand/or a nucleic acid encoding a dsRNA can be present within thebacterial genome, e.g. the nucleic acids can be incorporated into thegenome. In some embodiments, a nucleic acid encoding a siRNA-bindingpolypeptide and/or a nucleic acid encoding a dsRNA can be present withina vector. In some embodiments, a nucleic acid encoding a siRNA-bindingpolypeptide and/or a nucleic acid encoding a dsRNA can be present withinportions of the same vector. In some embodiments, the nucleic acidsencoding the siRNA-binding polypeptide and the dsRNA can be presentwithin portions of separate vectors.

The term “vector”, as used herein, refers to a nucleic acid constructdesigned for delivery to a host cell or transfer between different hostcells. As used herein, a vector can be viral or non-viral. Many vectorsuseful for transferring exogenous genes into target cells are available,e.g. the vectors may be episomal, e.g., plasmids, virus derived vectorsor may be integrated into the target cell genome, through homologousrecombination or random integration. In some embodiments, a vector canbe an expression vector. As used herein, the term “expression vector”refers to a vector that has the ability to incorporate and expressheterologous nucleic acid fragments in a cell. An expression vector maycomprise additional elements, for example, the expression vector mayhave two replication systems, thus allowing it to be maintained in twoorganisms. The nucleic acid incorporated into the vector can beoperatively linked to an expression control sequence when the expressioncontrol sequence controls and regulates the transcription andtranslation of that polynucleotide sequence. In some embodiments, thedsRNA and the nucleic acid encoding the siRNA-binding polypeptide can bewithin the same operon. In some embodiments, the dsRNA and the nucleicacid encoding the siRNA-binding polypeptide can be within separateoperons.

In some embodiments, a siRNA-binding polypeptide and/or dsRNA encoded bya nucleic acid can be present within a portion of a plasmid. Plasmidvectors can include, but are not limited to, pBR322, pBR325, pACYC177,pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40,pBluescript II SK+/− or KS+/− (see “Stratagene Cloning Systems” Catalog(1993) from Stratagene, La Jolla, Calif., which is hereby incorporatedby reference), pQE, pIH821, pGEX, pET series (see Studier et. al., “Useof T7 RNA Polymerase to Direct Expression of Cloned Genes,” GeneExpression Technology, vol. 185 (1990), which is hereby incorporated byreference in its entirety).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain a transgenic gene in place of non-essential viral genes. Thevector and/or particle may be utilized for the purpose of transferringany nucleic acids into cells either in vitro or in vivo. Numerous viralvectors are known in the art and can be used as carriers of a nucleicacid into a cell, e.g. lambda vector system gt11, gt WES.tB, Charon 4.

In accordance with the methods and compositions described herein, siRNAsspecific for the target RNA can be produced in a bacterial cell whenboth the dsRNA and the siRNA-binding polypeptide are present and/orexpressed. In some embodiments, the dsRNA and/or the siRNA-bindingpolypeptide can be constitutively expressed. In some embodiments,nucleic acids encoding the dsRNA and/or the siRNA-binding polypeptidecan be operably linked to a constitutive promoter. In some embodiments,the dsRNA and/or the siRNA-binding polypeptide can be induciblyexpressed. In some embodiments, nucleic acids encoding the dsRNA and/orthe siRNA-binding polypeptide can be operably linked to an induciblepromoter.

As described herein, an “inducible promoter” is one that ischaracterized by initiating or enhancing transcriptional activity whenin the presence of, influenced by, or contacted by an inducer orinducing agent than when not in the presence of, under the influence of,or in contact with the inducer or inducing agent. An “inducer” or“inducing agent” may be endogenous, or a normally exogenous compound orprotein that is administered in such a way as to be active in inducingtranscriptional activity from the inducible promoter. In someembodiments, the inducer or inducing agent, e.g., a chemical, a compoundor a protein, can itself be the result of transcription or expression ofa nucleic acid sequence (e.g., an inducer can be a transcriptionalrepressor protein), which itself may be under the control or aninducible promoter. Non-limiting examples of inducible promoters includebut are not limited to, the lac operon promoter, a nitrogen-sensitivepromoter, an IPTG-inducible promoter, a salt-inducible promoter, andtetracycline, steroid-responsive promoters, rapamycin responsivepromoters and the like. Inducible promoters for use in prokaryoticsystems are well known in the art, see, e.g. the beta.-lactamase andlactose promoter systems (Chang et al., Nature, 275: 615 (1978, which isincorporated herein by reference); Goeddel et al., Nature, 281: 544(1979), which is incorporated herein by reference), the arabinosepromoter system, including the araBAD promoter (Guzman et al., J.Bacteriol., 174: 7716-7728 (1 992), which is incorporated herein byreference; Guzman et al., J. Bacteriol., 177: 4121-4130 (1995), which isincorporated herein by reference; Siegele and Hu, Proc. Natl. Acad. Sci.USA, 94: 8168-8172 (1997), which is incorporated herein by reference),the rhamnose promoter (Haldimann et al., S. Bacteriol., 180: 12774286(1998), which is incorporated herein by reference), the alkalinephosphatase promoter, tryptophan (trp) promoter system (Goeddel, NucleicAcids Res., 8: 4057 (1980), which is incorporated herein by reference),the P_(LtetO-1) and P_(lac/are-1) promoters (Lutz and Bujard, NucleicAcids Res., 25: 1203-1210 (1997), which is incorporated herein byreference), and hybrid promoters such as the tac promoter. deBoer etal., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983), which is incorporatedherein by reference.

An inducible promoter useful in the methods and systems as disclosedherein can be induced by one or more physiological conditions, such aschanges in pH, temperature, radiation, osmotic pressure, salinegradients, cell surface binding, and the concentration of one or moreextrinsic or intrinsic inducing agents. The extrinsic inducer orinducing agent may comprise amino acids and amino acid analogs,saccharides and polysaccharides, nucleic acids, protein transcriptionalactivators and repressors, cytokines, toxins, petroleum-based compounds,metal containing compounds, salts, ions, enzyme substrate analogs,hormones, and combinations thereof. In specific embodiments, theinducible promoter is activated or repressed in response to a change ofan environmental condition, such as the change in concentration of achemical, metal, temperature, radiation, nutrient or change in pH. Thus,an inducible promoter useful in the methods and systems as disclosedherein can be a phage inducible promoter, nutrient inducible promoter,temperature inducible promoter, radiation inducible promoter, metalinducible promoter, hormone inducible promoter, steroid induciblepromoter, and/or hybrids and combinations thereof. Appropriateenvironmental inducers can include, but are not limited to, exposure toheat (i.e., thermal pulses or constant heat exposure), various steroidalcompounds, divalent cations (including Cu2+ and Zn2+), galactose,tetracycline, IPTG (isopropyl-β-D thiogalactoside), as well as othernaturally occurring and synthetic inducing agents and gratuitousinducers.

Inducible promoters useful in the methods and systems as disclosedherein also include those that are repressed by “transcriptionalrepressors” that are subject to inactivation by the action ofenvironmental, external agents, or the product of another gene. Suchinducible promoters may also be termed “repressible promoters” where itis required to distinguish between other types of promoters in a givenmodule or component of the biological switch converters describedherein. Preferred repressors for use in the present invention aresensitive to inactivation by physiologically benign agent. Thus, where alac repressor protein is used to control the expression of a promotersequence that has been engineered to contain a lacO operator sequence,treatment of the host cell with IPTG will cause the dissociation of thelac repressor from the engineered promoter containing a lacO operatorsequence and allow transcription to occur. Similarly, where a tetrepressor is used to control the expression of a promoter sequence thathas been engineered to contain a tetO operator sequence, treatment ofthe host cell with tetracycline will cause the dissociation of the tetrepressor from the engineered promoter and allow transcription of thesequence downstream of the engineered promoter to occur.

A bacterial cell of the methods and compositions described herein can beany of any species. Preferably, the bacterial cells are of a speciesand/or strain which is amenable to culture and genetic manipulation. Insome embodiments, the bacterial cell can be a gram-positive bacterialcell. In some embodiments, the bacterial cell can be a gram-negativebacterial cell. In some embodiments, the parental strain of thebacterial cell of the technology described herein can be a strainoptimized for protein expression. Non-limiting examples of bacterialspecies and strains suitable for use in the present technologies includeEscherichia coli, E. coli BL21, E. coli Tuner, E. coli Rosetta, E. coliJM101, and derivatives of any of the foregoing. Bacterial strains forprotein expression are commercially available, e.g. EXPRESS™ CompetentE. coli (Cat. No. C2523; New England Biosciences; Ipswich, Mass.).

A dsRNA comprising a nucleic acid sequence substantially complementaryto a target RNA can be processed to create siRNA molecules by asiRNA-generating enzyme (e.g. RNAse III) present within the bacterialcell. In some embodiments, the bacterial cell can be a cell whichexpresses a siRNA-generating polypeptide. In some embodiments, thebacterial cell can be a cell which overexpresses a siRNA-generatingpolypeptide. As used herein, a “siRNA-generating polypeptide” refers toan enzyme with RNase activity which can cleave dsRNA in such a way thatsiRNAs result. In some embodiments, the siRNA-generating polypeptide canbe an RNaseIII polypeptide. As used herein the term “RNaseIIIpolypeptide” refers to a eukaryotic class I RNase III, e.g. E. coliRNaseIII (NCBI Gene ID: 947033; SEQ ID NO: 2). siRNA-generatingpolypeptides can be mutants, variants, homologues, or functionalfragments of wildtype siRNA-generating polypeptides which retain atleast 50% of the siRNA generating activity of the wildtype, e.g. atleast 50%, at least 60%, at least 70%, at least 80%, at least 90% ormore of the wildtype activity. In some embodiments, the siRNA-generatingenzyme can be endogenous to the bacterial cell. In some embodiments, thesiRNA-generating enzyme can be exogenous to the bacterial cell.

In some embodiments, a cell can comprise a mutation and/or transgenewhich enhances the expression and/or activity of a siRNA-generatingpolypeptide. By way of non-limiting example, a cell can comprise amutation in the endogenous RNaseIII promoter which increases expression,or a cell can comprise a transgenic (e.g. exogenous) construct with anRNaseIII gene under the control of a strong constitutive or induciblepromoter, or a cell can comprise a nucleic acid encoding a polypeptidewhich increases the activity and/or expression of RNaseIII, e.g. the T4polynucleotide kinase/phosphatase (PNK) (see Durand et al. PNAS 2012109:7073-8; which is incorporated by reference herein in its entirety).In some embodiments, a cell can express an ectopic level and/or amountof a siRNA-generating polypeptide (e.g. RNaseIII). As used herein,“ectopic” refers to a substance that is found in an unusual locationand/or amount. An ectopic substance can be one that is normally found ina given cell, but at a lower amount and/or at a different time.

In one aspect, the technology described herein relates to a method ofproducing one or more siRNA species which can inhibit the expression ofa target RNA, the method comprising culturing a bacterial cellcomprising at least a siRNA-binding polypeptide and a dsRNA wherein thedsRNA comprises a nucleic acid sequence substantially complementary to atarget RNA under conditions suitable for the production of siRNAs. Asused herein, the term “conditions suitable for the production of siRNAs”refers to conditions under which a siRNA-generating enzyme within abacterial cell cleaves the dsRNA in the presence of a siRNA-bindingpolypeptide. In embodiments wherein one or more of the dsRNA and thesiRNA-binding polypeptide are encoded by nucleic acids, conditionssuitable for the production of siRNAs can include conditions under whichthe cell will express (i.e. transcribe and, in some cases, translate)the dsRNA and/or the siRNA-binding polypeptide from the nucleic acid.The precise conditions will vary depending on the exact identity of thebacterial cell, the presence of other exogenous DNA or mutations, andwhether or not a nucleic acid encoding a dsRNA and/or siRNA-bindingpolypeptide is operably linked to an inducible or constitutive promoter.In some embodiments, wherein the nucleic acid(s) encoding a dsRNA and/orsiRNA-binding polypeptide are operably linked to inducible promoters,conditions suitable for the production of siRNAs can include conditionswhich induce expression from the inducible promoter, e.g. permissivetemperatures and/or the presence of compounds which induce expressionfrom the inducible promoter. In some embodiments, conditions suitablefor the production of siRNAs can include conditions which encourageexponential growth of the bacterial cells. By way of non-limitingexample conditions suitable for the production of siRNAs in E. coli T7Express Iq (NEB) can include LB broth, Lennox (BD) at 37° C. withshaking at 250 rpm and appropriate antibiotics.

In some embodiments, a method of producing one or more siRNA species canfurther comprise isolating the siRNA-binding polypeptide and eluting thesiRNAs bound to the siRNA-binding polypeptide. In some embodiments, thesiRNA-binding polypeptide can be isolated via a purification tag asdescribed elsewhere herein.

In some embodiments, the siRNAs bound to a siRNA-binding polypeptide canbe eluted from the isolated siRNA-binding polypeptide. Methods ofeluting nucleic acids from proteins are well known in the art. By way ofnon-limiting example, siRNAs can be eluted from a siRNA-bindingpolypeptide by contacting the polypeptide-siRNA complex with a solutioncomprising 0.5% SDS for 10 min at room temperature with rotation. Thesolution can then be collected and passed through a 0.22 μm centrifugefilter (Corning).

In some embodiments, the siRNAs eluted from a siRNA-binding polypeptidecan be further purified. Methods of nucleic acid purification are wellknown in the art and include, but are not limited to anion exchangeHPLC, PAGE purification, desalting, and filtration. See, e.g. Gjerde etal. “RNA Purification and Analysis” Wiley-VCH; 2009 and Farrell et al.“RNA Methodologies” 4^(th) Ed., Academic Press; 2010. In someembodiments, the siRNAs eluted from a siRNA-binding polypeptide can befurther purified by HPLC.

In some embodiments, the siRNAs can be isolated from the totality of thecell contents without first isolating the siRNAs bound to siRNA-bindingpolypeptides. Methods of purifying RNA molecules are well known in theart, as described above, and any method or combination of methods knownin the art can be used to isolate and/or purify the siRNAs producedaccording to the methods described herein.

In some embodiments, the methods described herein can further comprisecontacting the bacterial cell with one or more modified nucleotidesbefore or during the culturing step, thereby causing one or moremodified nucleotides to be incorporated into the siRNA(s) of thepresently described technologies. A modified nucleotide can be anynucleotide other than adenine “A”, guanine “G”, uracil “U”, or cytosine“C”. Such modified nucleotides include nucleotides which contains amodified sugar moiety, a modified phosphate moiety and/or a modifiednucleobase. A modified nucleotide residue or a derivative or analog of anatural nucleotide are also useful. Examples of modified residues,derivatives or analogues include, but are not limited to, aminoallylUTP, pseudo-UTP, 5-I-UTP, 5-I-CTP, 5-Br-UTP, alpha-S ATP, alpha-S CTP,alpha-S GTP, alpha-S UTP, 4-thio UTP, 2-thio-CTP, 2′NH2 UTP, 2′NH2 CTP,and 2′F UTP. Such modified nucleotides include, but are not limited to,aminoallyl uridine, pseudo-uridine, 5-I-uridine, 5-I-cytidine,5-Br-uridine, alpha-S adenosine, alpha-S cytidine, alpha-S guanosine,alpha-S uridine, 4-thio uridine, 2-thio-cytidine, 2′NH2 uridine, 2′NH2cytidine, and 2′ F uridine, including the free pho (NTP) RNA moleculesas well as all other useful forms of the nucleotides. Furthernon-limiting examples of modified nucleotides can includeribonucleotides having a 2′-O-methyl (TOme), 2′-deoxy-2′fluoro,2′-deoxy, 5-C-methyl, 2′-methoxyethyl, 4′-thio, 2′-amino, or 2′-C-allylgroup, locked nucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-methoxyethoxy (MOE)nucleotides, 2′-methylthio-ethyl nucleotides, 2′-deoxy-2′-fluoronucleotides, 2′-deoxy-2′-chloro nucleotides, and 2′-azido nucleotides.),nucleotides having a nucleotide base analog such as, for example,C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azolecarboxamides, and nitroazole derivatives such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole. Modification of thesugar moiety can include, but is not limited to, replacement of theribose, ring with a hexose, cyclopentyl or cyclohexyl ring.Alternatively, the D-ribose ring of a naturally-occurring nucleic acidcan be replaced with an L-ribose ring or the (3-anomer of a naturallyoccurring nucleic acid can be replaced with the a-anomer. Modifiedphosphate moieties can include phosphorothioates, phosphomdithioates,methyl phosphonates, alkylphosphonates, alkylphosphonothioates, methylphosphates, phosphoramidates, and the like, or combinations thereof.Oligonucleotides which comprise such modified phosphate linkages canhave improved properties when compared to corresponding oligonucleotidescomprising only phosphate diester linkages, e.g. increased resistance todegradation by nucleases. Modified nucleobases include 7-deazaguanine,7-deaza-8-azaguanine, 5-propynylcytosine, 5-propynyluricil,7-deazaadenine, 7-deaza-8azaadenine, 7-deaza-6-oxopurine, 6-oxopurine,3-deazaadenosine, 2-oxo-5-methylpyrimidine,2-oxo-4-methylfhio-5methylpyrimidine,2-thiocarbonyl-4-oxo-5-methylpyrimidine, 4-oxo-5-methylpyrimidine,2-aminopurine, 5-fluorouricil, 2,6-diaminopurine, 8-aminopurine,4triazolo-5-methylthymine, and 4-triazolo-5-mefhyluricil. Modifiednucleobases can also include abasic moieties. Additional non-limitingexamples of modified nucleotides include biotinylated nucleotides,amine-modified nucleotides, alkylated nucleotides, fluorophore-labelednucleotides, radiolabeled nucleotides, phosphorothioates,phosphoramidites, phosphites, ring atommodified derivatives and thelike. In some embodiments, a modified nucleotide can be a G-clampnucleotide. A G-clamp nucleotide refers to a modified cytosine analogwherein the modifications confer the ability to hydrogen bond bothWatson-Crick and Hoogsteen faces of a complementary guanine nucleotidewithin a duplex (see, e.g., Lin et al., J Am. Chem. Soc., 120:8531-8532(1998); which is incorporated herein by reference in its entirety. Insome embodiments, a modified nucleotide can comprise multiplemodifications. In some embodiments, a cell can be contacted with anycombination of modified nucleotides.

In one aspect, the technology described herein relates to a library ofsiRNA species, the library comprising a plurality of clonal bacterialcell populations; wherein each clonal population comprises bacterialcells as described herein. In some embodiments, the bacterial cells cancomprise at least a siRNA-binding polypeptide and a dsRNA; wherein thedsRNA comprises a nucleic acid sequence substantially complementary to atarget RNA. In some embodiments, wherein a bacterial cell comprisesdsRNAs specific for a single target RNA, the clonal bacterial populationcomprising that cell can comprise a population of siRNAs which willspecifically bind to the single target RNA and/or which willspecifically silence the expression of the target RNA.

In one aspect, the technology described herein relates to a library ofsiRNA species, the library comprising a plurality of populations ofsiRNAs; wherein each population of siRNAs is obtained according to themethods described herein. As used herein, a “population of siRNAs”refers to two or more siRNAs, wherein at least two of the siRNAscomprise non-identical sequences, but wherein the two or more siRNAseach comprise a nucleic acid sequence substantially complementary to thesame target RNA. For example, a population of siRNAs can comprise twomore siRNA species. In some embodiments, a population of siRNAs can begenerated from a single dsRNA comprising a nucleic acid sequencesubstantially complementary to a target RNA. In some embodiments, apopulation of siRNAs can bind to a single target RNA and/or canspecifically silence the expression of the target RNA. A population ofsiRNAs can be present within a bacterial cell or isolated from abacterial cell.

Methods of creating bacterial libraries, and/or libraries of compoundsisolated from bacterial cells are well known in the art. By way ofnon-limiting example, a bacterial cell library can be in the form of aplurality of multi-well plates, with each well of a plate comprising aclonal bacterial population. The clonal bacterial populations can beprovided in media or in glycerol stocks. In some embodiments, a librarycan comprise multiple wells which comprise identical clonal populations,i.e. a clonal population can appear multiple times in a library. In someembodiments, a library can comprise a plurality of multi-well plates,with each well of a plate comprising one or more siRNA species (e.g. asiRNA species or a population of siRNA species) isolated from one ormore clonal bacterial populations. Methods of isolating nucleic acidsfrom bacterial cells are well known in the art and examples aredescribed elsewhere herein. In some embodiments, libraries can becreated using automated and/or high-throughput methods, e.g. roboticcolony-picking.

In some embodiments, a library can comprise pooled samples, e.g.multiple clonal bacterial populations, multiple isolated siRNAs, ormultiple isolated populations of siRNA species can be pooled so that asmaller number of samples must be initially screened. The individualcomponents of a “positive” pooled can be subsequently screenedseparately.

In some embodiments, a library can comprise 10 or more pools of,populations of, and/or individual siRNA species (e.g. isolated orpresent within bacterial cells), e.g. 10 or more, 100 or more, 1,000 ormore, 10,000 or more, or 100,000 or more pools of, populations of,and/or individual siRNA species.

In some embodiments, a library can comprise a plurality of populationsof siRNAs, wherein each population of siRNAs can silence at least onetarget RNA of a target set. A target set of RNAs can comprise, e.g. thetranscriptome of a cell, the transcriptome of an organism, thetranscriptome of a cell and/or organism in a specific state (e.g. adiseased organism or an organism at a specific stage of development) ora subtractive transcriptome (e.g. all the transcripts present in a cellunder one condition but which are not present in the cell in a secondcondition).

In one aspect, the technology described herein relates to vectors whichenable the use of the methods and compositions described herein. In someembodiments, the vector can be an expression vector. In someembodiments, the vector can be a plasmid. In some embodiments, a vectorfor use in the methods and compositions described herein can comprise(a) a nucleic acid encoding a siRNA-binding polypeptide and (b) a dsRNAcloning site. In some embodiments, a dsRNA cloning site further canfurther comprise a nucleic acid encoding a dsRNA, wherein the dsRNAcomprises a nucleic acid sequence substantially complementary to atarget RNA. As used herein, a “dsRNA cloning site” refers to a multiplecloning site comprising at least one restriction enzyme site and whichcan accept the insertion of nucleic acid sequence(s) comprising thesequence of both a sense and anti-sense strand of nucleic acid; whereinone strand is substantially complementary to the nucleic acid sequenceof a target RNA, such that a dsRNA will be encoded and can be expressed,e.g. a sequence inserted at the dsRNA cloning site will be operablylinked to a promoter as described herein. In some embodiments, a singlenucleic acid molecule can comprise the sequence of both the sense andanti-sense strand prior to insertion at the dsRNA cloning site. In someembodiments, a dsRNA cloning site can comprise a nucleic acid sequencewhich comprises sequences which can be cleaved by at least two differentrestriction enzymes.

In some embodiments, a dsRNA cloning site can comprise a nucleic acidsequence which comprises sequences which can be cleaved by at least fourdifferent restriction enzymes. In some embodiments, a dsRNA cloning sitecan comprise two multiple cloning sites separated by a nucleic acidsequence encoding a hairpin sequence; wherein each multiple cloning sitecomprises a nucleic acid sequence which comprises sequences which can becleaved by at least two different restriction enzymes. Methods ofcloning various dsRNA sequences into expression vectors, as well asexpression vectors which can be adapted for use as described herein, arewell known in the art, see, e.g. Schwab et al. 2006 Plant Cell18:1121-1133; Fraser. AfCS Reports 2004; Atayde et al. Mol BiochemParasitol 2012 184:55-8: Kruhn et al. Cell Cycle 2009 8:3349-3354; andTimmons et al. Gene 2001 263:103-112; which are incorporated byreference herein in their entireties.

In some embodiments, a vector for use in the methods and/or compositionsdescribed herein can comprise at least one constitutive promoteroperably linked to at least one of the siRNA-binding polypeptide or thedsRNA multiple cloning site. In some embodiments, a vector for use inthe methods and/or compositions described herein can comprise at leastone inducible promoter operably linked to at least one of thesiRNA-binding polypeptide or the dsRNA multiple cloning site.

Aspects of the technology described herein further relate to kitscomprising the compositions described herein and kits for practicing themethods described herein.

In some embodiments, the technology described herein relates to a kitcomprising a bacterial cell as described herein, e.g. a bacterial cellcomprising at least a siRNA-binding polypeptide and a dsRNA; wherein thedsRNA comprises a nucleic acid sequence substantially complementary to atarget RNA as described herein.

In some embodiments, the technology described herein relates to a vectorfor use in the methods and compositions of the present technology, asdescribed herein. In some embodiments, a kit for the production of oneor more species of siRNA can comprise a vector comprising (a) a nucleicacid encoding a siRNA-binding polypeptide and (b) a dsRNA cloning site.In some embodiments, a kit for the production of one or more species ofsiRNA can comprise two vectors; wherein the first vector comprises anucleic acid encoding a siRNA-binding polypeptide; and wherein thesecond vector comprises a dsRNA cloning site. In some embodiments, thedsRNA cloning site can further comprise a dsRNA; wherein the dsRNAcomprises a nucleic acid sequence substantially complementary to atarget RNA. In some embodiments, the kit can further comprise abacterial cell.

In some embodiments, a kit for the production of one or more species ofsiRNA can comprise a bacterial cell comprising a siRNA-bindingpolypeptide and a vector comprising a dsRNA cloning site. In someembodiments, the bacterial cell can comprise a nucleic acid encoding asiRNA-binding polypeptide. In some embodiments, the nucleic acidencoding a siRNA-binding polypeptide can be a part of an expressionvector, a plasmid, a naked nucleic acid, and/or the bacterial genome.

In some embodiments of a kit as described herein, the siRNA-bindingpolypeptide can comprise a purification tag. In some embodiments of akit as described herein, the siRNA-binding polypeptide can be encoded bya nucleic acid. In some embodiments of a kit as described herein, theDNA encoding at least one of the siRNA-binding polypeptide or the dsRNAcan be a portion of a vector. In some embodiments of a kit as describedherein at least one of the siRNA-binding polypeptide or the dsRNA can beconstitutively expressed. In some embodiments of a kit as describedherein, at least one of the siRNA-binding polypeptide or the dsRNA canbe inducibly expressed.

In some embodiments of a kit as described herein, the bacterial cell canexpress a siRNA-generating polypeptide. In some embodiments of a kit asdescribed herein, the cell can be an Escherichia coli cell.

In some embodiments, the technology described herein relates to a kitcomprising a library of siRNA species as described herein.

In some embodiments, the compositions and methods described herein canbe used to test the efficacy of one or more siRNA species, and/or forthe screening of a siRNA library.

In some embodiments, the efficacy of one or more siRNA species can beassessed in cultured mammalian cells. Methods of targeting mammaliancells with inhibitory RNAs via bacterial invasion are known in the art,see, e.g. Zhao et al. Nature Methods 2005 2:967-973; which isincorporated by reference herein in its entirety. In some embodiments, abacterial cell for use in such an assay can comprise a polypeptide ornucleic acid encoding a polypeptide which can bind to a mammalian cellsurface receptor, (e.g. the invasin (inv) gene of Yersiniapsuedotuberculosis which binds the integrin receptor of mammalian cells.In some embodiments, a bacterial cell for use in such an assay or screencan comprise a mutation reducing the ability of the cell to synthesizeor maintain the cell wall, (e.g. deletion of the asd gene of E. coli,thereby rendering the cell a diaminopimelic acid (DAP) auxotroph).Reducing the ability of the cell to synthesize or maintain the cell wallcan make the cell susceptible to lysis or degradation after it enters amammalian cell, thereby releasing inhibitory RNAs (e.g. in the methodsdescribed herein, siRNAs) into the mammalian cell.

In some embodiments, the efficacy of one or more siRNA species can beassessed in vivo in C. elegans. dsRNAs readily cross cell membranes inC. elegans, and a number of protocols are known for conducting RNAi inC. elegans, including bacterial feeding assays (see, e.g. Timmons, L.,and A. Fire. Nature 1998 395:854 and Lehner et al. Protocol Exchange2006 159; which are incorporated by reference herein in theirentireties.

In some embodiments, the efficacy of one or more siRNA species can beassessed by first isolating the one or more species of siRNA from thebacterial cells and then contacting a cell and/or organism with the oneor more species of siRNA. Methods of introducing ribonucleic acids, andin particular, ribonucleic acids which cause RNAi into various cells andorganisms are well known in the art (see, e.g. Sioud, M. “siRNA andmiRNA Gene Silencing” Humana Press: 2011; “Gene Silencing by RNAInterference” Sohail, M. ed. CRC Press: 2004: each of which areincorporated by reference herein in their entireties). Examples of cellsand/or organisms suitable for use in such methods include cultured cells(e.g. mammalian cells or human cells), primary cells, diseased cells(e.g. cancerous cells), C. elegans, and Danio rerio.

The efficacy of one or more siRNA species can be assessed by screens,selections, and/or by assays. High throughput methods of screening siRNAlibraries are known in the art, e.g. phenotype screens, automated celland worm processing, etc. The appropriate method of determining theefficacy of one or more siRNA species can be dependent upon the natureof the target RNA, e.g. siRNA species specific for target RNAs whichcontrol reproduction in C. elegans can be screened by examining the rateand success of reproduction of worms in the presence of the siRNAs.

In some embodiments, libraries of siRNA species as described herein,comprising siRNA species targeting a number of different target RNAs canbe used in phenotypic screens to identify target RNAs associated with aparticular phenotype (e.g. siRNAs which perturb a particulardevelopmental process or which slow the progression of a disease).Phenotypic screens can comprise the assays described above fordetermining efficacy, e.g. mammalian cell invasion assays. In someembodiments, phenotypic screens can involve high-throughput assays.

In one aspect, described herein is a therapeutic agent comprising asiRNA species or population of siRNA species isolated from a bacterialcell as described herein and/or produced according to the methodsdescribed herein. According to the methods described herein, a dsRNAcomprising a nucleic acid substantially complementary to a target RNAcan be provided to a bacterial cell herein, and a siRNA species and/orpopulation of siRNA species which can be used to reduce the expressionof the corresponding target RNA can be produced. In some embodiments,target RNAs can be disease-associated RNAs, i.e. RNAs whoseoverexpression is associated with the cause, progression, or maintenanceof a disease state, e.g. oncogenes. In some embodiments, target RNAs canbe RNAs originating from a pathogenic organism, e.g. the target RNAs cancomprise sequences of viral, bacterial, fungal, and/or parasitic origin.In some embodiments, target RNAs can be viral RNAs and/or RNAs producedfrom viral genomic material. In some embodiments, a siRNA species and/orpopulation of siRNA species which can be used to reduce the expressionof the target RNA can be produced according to the methods describedherein and administered to a subject in need of a reduction of the levelof expression of the target RNA. In some embodiments, a single siRNAspecies can be administered. In some embodiments, a population of siRNAspecies can be administered. As demonstrated in the Examples herein, apopulation of siRNA species can have increased efficacy and a lowerlikelihood of off-target effects as compared to a single siRNA species.In some embodiments, multiple populations of siRNA species can beadministered, i.e. multiple target RNAs can be silenced. In someembodiments, the technology described herein relates to a pharmaceuticalcomposition comprising a bacterial cell, siRNA species, and/orpopulation of siRNA species according to the methods and compositionsdescribed herein. In some embodiments, the technology described hereinrelates to the use of a bacterial cell, siRNA species, or population ofsiRNA species according to the methods and compositions described hereinin the manufacture of a medicament. Methods of preparing medicamentscomprising RNA molecules, e.g. siRNAs, are known in the art, (see e.g.Oh and Park. Advanced Drug Delivery Reviews. 2009 61:850-62; which isincorporated by reference herein in its entirety).

It is contemplated that the siRNA technology described herein, as wellas the methods and compositions relating thereto, can be applied to genesilencing applications in any cell and/or organism comprising siRNAmachinery. Non-limiting examples include gene silencing applicationshumans, non-human animals, livestock species, insects (e.g. honeybees),plants, crop plants, etc. In some embodiments, the gene silencing can befor therapeutic purposes. In some embodiments, the gene silencing can befor agricultural purposes, e.g. to treat agricultural diseases inanimals and/or crops or to increase yields in animals and/or crops.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A bacterial cell comprising a siRNA-binding polypeptide and a        dsRNA comprising a nucleic acid sequence substantially        complementary to a target RNA.    -   2. The bacterial cell of paragraph 1, wherein the siRNA-binding        polypeptide comprises a purification tag.    -   3. The bacterial cell of any of paragraphs 1-2, wherein the        siRNA-binding polypeptide is encoded by a nucleic acid.    -   4. The bacterial cell of any of paragraphs 1-3, wherein the        siRNA-binding polypeptide is selected from the group consisting        of:        -   p19 polypeptide; tombusvirus p19 polypeptide; B2            polypeptide; HC-Pro polypeptide;        -   p38 polypeptide; p122 polypeptide; p130 polypeptide; p21            polypeptide; p1b polypeptide; and NS3 polypeptide.    -   5. The bacterial cell of any of paragraphs 1-4, wherein the        dsRNA is greater than 21 nucleotides in length.    -   6. The bacterial cell of any of paragraphs 1-5, wherein the        dsRNA is a hairpin RNA.    -   7. The bacterial cell of any of paragraphs 1-6, wherein the        bacterial cell expresses an RNase III polypeptide.    -   8. The bacterial cell of any of paragraphs 1-7, wherein the        bacterial cell expresses an RNase III polypeptide encoded by an        exogenous nucleic acid sequence.    -   9. The bacterial cell of any of paragraphs 1-8, wherein the        bacterial cell is an Escherichia coli cell.    -   10. The bacterial cell of any of paragraphs 1-9, wherein at        least one of the siRNA-binding polypeptide and the dsRNA are        constitutively expressed.    -   11. The bacterial cell of any of paragraphs 1-10, wherein at        least one of the siRNA-binding polypeptide and the dsRNA are        inducibly expressed.    -   12. The bacterial cell of any of paragraphs 1-11, wherein the        DNA encoding at least one of the siRNA-binding polypeptide or        the dsRNA is part of a plasmid.    -   13. The bacterial cell of any of paragraphs 1-12, wherein the        dsRNA comprises nucleic acid sequences substantially        complementary to a multiplicity of target RNAs.    -   14. A method of producing one or more siRNA species which can        inhibit the expression of a target RNA, the method comprising:        -   culturing a bacterial cell of any of paragraphs 1-13 under            conditions suitable for the production of siRNAs.    -   15. The method of paragraph 14, further comprising a second step        of isolating the siRNA-binding polypeptide and eluting the        siRNAs bound to the siRNA-binding polypeptide.    -   16. The method of any of paragraphs 14-15, further comprising        purifying the siRNAs eluted from the siRNA-binding polypeptide        by HPLC.    -   17. The method of any of paragraphs 14-16, further comprising        contacting the cell with one or more modified nucleotides before        or during the culturing step.    -   18. A pharmaceutical composition comprising a siRNA produced        according to the method of any of paragraphs 14-17.    -   19. The composition of paragraph 18, further comprising a        population of siRNA species.    -   20. A pharmaceutical composition comprising a siRNA isolated        from a bacterial cell of any of paragraphs 1-13.    -   21. The composition of paragraph 20, further comprising a        population of siRNA species.    -   22. The use of a siRNA produced according to the method of any        of paragraphs 14-17 in the production of a medicament.    -   23. The use of a siRNA isolated from a bacterial cell of any of        paragraphs 1-13 in the production of a medicament.    -   24. A vector comprising;        -   a nucleic acid encoding a siRNA-binding polypeptide; and        -   a dsRNA cloning site.    -   25. The vector of paragraph 24, wherein the dsRNA cloning site        comprises at least one restriction enzyme site and can accept        the insertion of at least one nucleic acid sequence such that a        dsRNA is encoded and can be expressed.    -   26. A vector comprising:        -   a nucleic acid encoding a siRNA-binding polypeptide; and        -   a dsRNA comprising a nucleic acid sequence substantially            complementary to a target RNA.    -   27. The vector of any of paragraphs 24-26, wherein the        siRNA-binding polypeptide is selected from the group consisting        of:        -   p19 polypeptide; tombusvirus p19 polypeptide; B2            polypeptide; HC-Pro polypeptide;        -   p38 polypeptide; p122 polypeptide; p130 polypeptide; p21            polypeptide; p1b polypeptide; and NS3 polypeptide.    -   28. The vector of any of paragraphs 24-27, wherein the vector is        a plasmid.    -   29. The vector of paragraph 28, wherein the plasmid further        comprises a bacterial origin of replication.    -   30. A library of siRNA species, the library comprising:        -   a plurality of clonal bacterial cell populations;        -   wherein each clonal population is comprises bacterial cells            of any of paragraphs 1-13.    -   31. A library of siRNA species, the library comprising:        -   a plurality of populations of siRNAs;        -   wherein each population of siRNAs is obtained according to            the methods of any of paragraphs 14-17.    -   32. The library of paragraph 31, wherein each population of        siRNAs binds to a single target        -   RNA.    -   33. A kit comprising a bacterial cell of any of paragraphs 1-13.    -   34. A kit for the production of one or more species of siRNA,        the kit comprising;        -   a bacterial cell comprising an siRNA-binding polypeptide;            and        -   at least one vector comprising a dsRNA cloning site.    -   35. A kit for the production of one or more species of siRNA,        the kit comprising:        -   a bacterial cell comprising an siRNA-binding polypeptide;            and        -   at least one vector comprising a dsRNA comprising a nucleic            acid sequence substantially complementary to a target RNA.    -   36. A kit comprising the vector of any of paragraphs 24-29.    -   37. A kit for the production of one or more species of siRNA,        the kit comprising two vectors;        -   wherein the first vector comprises a nucleic acid encoding a            siRNA-binding polypeptide; and        -   wherein the second vector comprises a dsRNA cloning site.    -   38. A kit for the production of one or more species of siRNA,        the kit comprising two plasmids;        -   wherein the first vector comprises a nucleic acid encoding a            siRNA-binding polypeptide; and        -   wherein the second vector comprises a dsRNA comprising a            nucleic acid sequence substantially complementary to a            target RNA.    -   39. The kit of any of paragraphs 33-38, wherein at least one        vector is a plasmid.    -   40. The kit of paragraph 39, wherein the plasmid further        comprises a bacterial origin of replication.    -   41. The kit of any of paragraphs 33-40, wherein the kit further        comprises a bacterial cell.    -   42. A kit for the production of one or more species of siRNA,        the kit comprising;        -   a bacterial cell comprising a nucleic acid encoding a            siRNA-binding polypeptide; and        -   a vector comprising a dsRNA cloning site.    -   43. A kit for the production of one or more species of siRNA,        the kit comprising;        -   a bacterial cell comprising a nucleic acid encoding a            siRNA-binding polypeptide; and        -   a vector comprising a dsRNA comprising a nucleic acid            sequence substantially complementary to a target RNA.    -   44. The kit of any of paragraphs 33-43, wherein the        siRNA-binding polypeptide comprises a purification tag.    -   45. The kit of any of paragraphs 33-44, wherein the        siRNA-binding polypeptide is encoded by a nucleic acid.    -   46. The kit of any of paragraphs 41-45, wherein the bacterial        cell expresses an RNase III polypeptide.    -   47. The kit of any of paragraphs 41-46, wherein the cell is an        Escherichia coli cell.    -   48. The kit of any of paragraphs 33-47, wherein at least one of        the siRNA-binding polypeptide and the dsRNA are operably linked        to a constitutive promoter.    -   49. The kit of any of paragraphs 33-48, wherein at least one of        the siRNA-binding polypeptide and the dsRNA are operably linked        to an inducible promoter.    -   50. The kit of any of paragraphs 33-49, wherein the DNA encoding        at least one of the siRNA-binding polypeptide or the dsRNA is        part of a plasmid.    -   51. A kit comprising the library of any of paragraphs 30-32.

EXAMPLES

RNA interference (RNAi) by double-stranded (ds) small interfering RNAs(siRNA) suppresses gene expression by inducing the degradation of mRNAsbearing complementary sequences^(1,2). Endogenous siRNAs (perfectlypaired dsRNAs ˜21-25 nt in length) play an important role in hostdefense against RNA viruses and in transcriptional gene silencing inplants and may have similar functions in other eukaryotes³. Transfectionof synthetic siRNAs into eukaryotic cells^(4,5) to silence genes hasbecome an indispensable tool to investigate gene function, andsiRNA-based therapy is being developed to knockdown genes implicated indisease⁶. Although bacteria expressing sense and antisense sequences canbe fed to worms to knock down individual genes⁷, no one has used livingorganisms to produce highly active, purified siRNAs. Described herein isa method to produce highly potent siRNAs from E. coli ectopicallyexpressing p19, a siRNA binding protein, which stabilizes siRNA-likespecies generated by bacterial RNase III.

The most common method to make siRNA is chemical synthesis^(4,5).Effective siRNA sequences are predicted using computer algorithms.siRNAs can also be made from transcribed longer dsRNAs by in vitrobiochemical processing by RNase III family enzymes^(8,9). In the lattercase, the resulting siRNAs contain many sequences against one target,which sometimes can be more effective than any one sequence¹⁰, and poolsof siRNAs often have fewer off-target effects on genes bearing partiallycomplementary sequences¹¹. While gene knockdown by transfection ofsiRNAs is usually transient, short hairpin RNA construct, delivered byplasmid or lentivirus, is commonly used to achieve stable genesilencing.

p19, an RNAi suppressor protein encoded by the plant RNA virustombusvirus¹², selectively binds to ˜21 nt siRNAs, including thosetargeting the virus¹³. The p19 dimer binds to the 19 nt duplex region ofan siRNA in a sequence-independent manner^(14,15). It was originallyplanned to enrich for endogenous siRNAs in mammalian cells using p19coupled to magnetic beads¹⁵. As a negative control, p19 beads wereincubated with total RNA isolated from E. coli, an organism thatsupposedly lacks the RNAi machinery, that was transformed or not with apcDNA3.1+ plasmid in which p19 was cloned after the CMV immediate-earlypromoter. Surprisingly p19 beads pulled down ˜21 nt dsRNAs from RNA ofboth human T-cells (ACH2 cell line) and the transformed E. coli cells(FIG. 1A). Although the CMV promoter is mostly used for efficientexpression of genes in mammalian cells, E. coli harboring pcDNA3.1+plasmids encoding FLAG-tagged TREX1 or p19 gene expressed theirrespective proteins (FIG. 1B). When total RNA isolated from E. colitransformed with empty vector or vectors encoding p19 or TREX1 wasseparated on SYBR Gold-stained denaturing polyacrylamide gels, adistinctive ˜21 nt band was evident only in p19-expressing E. coli (FIG.1B). These data indicate that p19 protein expression may have stabilizeda cryptic siRNA-like RNA species in E. coli. In Listeria monocytogenes,a Gram-positive bacterium, expression of p19 also allowed the detectionof ˜21 nt small RNAs (FIGS. 5A-5B).

To determine if the small RNAs detected in E. coli depended onfunctional p19, RNA was isolated from E. coli expressing WT p19, or p19mutants that disrupted siRNA binding^(14,16) (FIG. 1C). The ˜21 nt dsRNAband was more prominent in bacteria expressing WT p19. ThussiRNA-binding to p19 promotes the accumulation of siRNA-like RNAs in E.coli. Next the nuclease responsible for making small RNAs was sought.The most likely candidate was RNase III, an ancestor of eukaryoticDicer, responsible for the final step of siRNA biogenesis¹⁷ . E. coliRNase III is known to generate siRNA-sized dsRNAs from longer dsRNAs invitro⁹. p19-expressing plasmids were used to transform two RNase IIImutant strains, rnc14¹⁸ and rnc38¹⁹ (FIG. 1D). In both mutant strains,p19 beads failed to pull down any visible small RNAs. Furthermorerestoration of RNase III expression in HT115(DE3), a rnc14 strain, alsorestored the p19-dependent small RNAs (FIG. 1E), providing support forthe hypothesis that RNase III is responsible for generating these smallRNAs in E. coli. Thus, accumulation of these bacterial small RNAsdepends on ectopic p19 and bacterial RNase III.

It was next asked whether small RNAs generated in p19-expressing E. colibehave like siRNAs and can be used for gene knockdown in mammaliancells. p19 was cloned into the pGEX-4T-1 plasmid to express a GST-p19fusion protein with a C-terminal His tag (FIG. 2A). A T7 promoterdriving expression of a hairpin RNA that contains a target sequence wasinserted immediately after. To develop the method, a hairpin wasdesigned that encoded full-length EGFP (EGFPFL). The expression of theGST-p19-His fusion protein and hairpin RNA were both induced by IPTG.The GST-p19-His protein was captured by Nickel (Ni) affinitychromatography and 0.5% SDS was used to selectively elute p19-bound RNAsthat were predominantly ˜21 nt long (FIGS. 2B and 6A-6D). Small RNAswere further purified from other longer RNAs by anion exchange HPLC. Toverify that these bacterial small RNAs are double-stranded, they weretreated with a variety of nucleases. Like chemically synthesized siRNAs,bacterial small RNAs were sensitive to RNase A, but were insensitive toenzymes that digest ssRNA or DNA (Xrn1, RNase T1, exonuclease T (Exo T),exonuclease I (Exo I), or DNase Turbo (FIG. 2C). Next bacterial smallRNAs, purified from E. coli expressing p19 and the EGFPFL hairpin andtransfected into HeLa cells stably expressing d1EGFP (HeLa-d1EGFP), wereloaded into the RNA-induced silencing complex (RISC) byimmunoprecipitation with a pan-Argonaute (Ago) antibody (FIG. 2D). RNAsthat precipitated with anti-Ago were ˜21 nt long and hybridized to anEGFP probe, but no small RNA precipitated with control mouse IgG. Thusbacterial small RNAs were similar to synthetic siRNA in chemicalcomposition and were incorporated into the RISC. These small RNAs werenamed ‘pro-siRNAs’ for prokaryotic siRNAs.

Since pro-siRNAs had properties of siRNAs, whether p19-captured EGFPpro-siRNAs induce gene knockdown was tested. qRT-PCR and flow cytometrywere used to compare mRNA and protein knockdown, respectively, of d1EGFPin HeLa-d1EGFP cells transfected with a synthetic EGFP siRNA orpro-siRNAs purified from E. coli expressing p19 and hairpins of eitherfull length EGFP (EGFPFL) or a 100 nt fragment that overlapped with theEGFP siRNA sequence (EGFP100). Both EGFPFL and EGFP100 pro-siRNAsknocked down EGFP expression more effectively than equimolarconcentrations of siRNA (FIGS. 2E and 7A). pro-siRNAs made from theplasmid without or with only half of the EGFP hairpin could not silenceEGFP effectively (FIG. 7B). As expected, silencing by pro-siRNA wasDicer-independent because EGFPFL pro-siRNA still functioned inDicer-deficient HCT116 cells²⁰ and recombinant Dicer protein did notfurther process pro-siRNAs in vitro (FIGS. 8A-8B).

To test the effectiveness of pro-siRNA knockdown of endogenous and viralgenes, we used convenient restriction sites to clone and expresshairpins from the coding regions of LMNA (which encodes two splicevariant products, lamin A and lamin C), PLK1, TP53 and HIV vif (viralinfectivity factor) and gag (capsid antigen) to purify pro-siRNAs. Theresulting hairpins contained 200-579 nt of each sense and antisensesequence (523 nt for LMNA, 299 nt for PLK1, 300 nt for TP53, 579 nt forvif200 and 500 nt for gag). The HPLC-purified pro-siRNAs for each genecontained a few different sized species that migrated close to the 21 ntmarker on both native and denaturing polyacrylamide gels (FIG. 2F). ForLMNA and PLK1 pro-siRNAs, a minor RNA band migrated at ˜25 nt. Next theextent of knockdown of endogenous genes (LMNA, TP53, PLK1) by pro-siRNAsand commercially available siRNAs (LMNA and TP53 siRNAs were from asingle sequence; PLK1 siRNAs were a pool of 4 siRNAs and were chemicallymodified by proprietary methods for enhanced stability and reducedoff-target effects²¹) in HeLa-d1EGFP and HCT116 cells were compared. Theextent of gene knockdown was similar between siRNA and pro-siRNAtransfected at 4 nM (FIG. 3A). Since knocking down PLK1 causes death ofdividing cells²², viable cells were counted for 3 d followingtransfection with PLK1 or control siRNAs and pro-siRNAs (FIG. 3B). Tomore closely evaluate the potency of pro-siRNAs, dose responseexperiments comparing transfection of pro-siRNAs (0.2, 2, 20 nM)targeting LMNA, TP53 and PLK1 with five commercial siRNAs for each gene(four siRNAs from Dharmacon, of which the PLK1 siRNAs were chemicallymodified for enhanced RISC uptake or stability by proprietary methods,and one siRNA sequence chosen based on published effectiveness) wereperformed (FIG. 9). The potency of the commercial siRNAs varied, as bestevaluated at the lowest concentration. The pro-siRNAs, whose sequenceswere not optimized, achieved similar gene knockdown as the commerciallyoptimized siRNAs. At a concentration of 2 nM, each pro-siRNA achievedknockdown of ˜90%. Because siRNA design algorithms are imperfect,identifying a potent siRNA usually requires test of several sequenceswhich could be costly and time consuming. pro-siRNAs might circumventthe need to test multiple sequences to identify a single potent siRNA.

To examine potential toxicity of pro-siRNAs, growth was compared over 3d in HeLa-d1GFP and HCT116 cells after transfection with either anegative control siRNA or EGFP pro-siRNA (FIG. 3B). Their growth curveswere not significantly different. To compare the effectiveness of geneknockdown by pro-siRNAs and siRNAs, cell proliferation was examinedafter knocking down PLK1, which kills dividing cells²³. PLK1 siRNAs andpro-siRNAs both dramatically reduced viability with indistinguishablekinetics.

As another test of pro-siRNA function, the effect of knocking down theHIV accessory gene vif on in vitro propagation of HIV infection²³ wasexamined. vif, which targets the host restriction factor APOBEC3G forubiquitylation and degradation, is not needed for the initial round ofHIV replication, but is required to spread the infection to new cells bypreventing APOBEC3G packaging into budding virions. The efficacy of thepro-siRNAs was compared with two validated siRNAs^(23,24). As expected,siRNAs and pro-siRNAs targeting vif did not alter the percentage ofinitially infected HeLa-CD4 cells (data not shown), but did suppress vifgene expression and inhibit subsequent rounds of infection, assessed inthe TZM-bl luciferase reporter cell line (FIG. 3D). Transfection of vifpro-siRNAs resulted in much lower levels of vif mRNA in HeLa-CD4 cellsand HIV tat-driven luciferase activity, compared to transfection witheither or both vif siRNAs. Thus vif pro-siRNAs were superior topreviously used siRNAs in inhibiting HIV spread in vitro.

One major obstacle to using RNAi to suppress HIV or other viruses issequence diversity. Because pro-siRNAs target many sequences within agene, pro-siRNAs directed against a viral gene can have broader activityagainst diverse viral strains than siRNAs and can also be less likely togenerate siRNA-resistant mutants. Previous attempts to identify an siRNAagainst HIV-1 clade B gag that could inhibit viral isolates from otherclades were unsuccessful²⁴. A sequence that protected against infectionwith all clade B viruses was tested, but no single sequences which werewell enough conserved were found that also protected against otherclades. To investigate whether pro-siRNAs might have broader activitythan the best clade B sequence, gag pro-siRNAs were engineered usinghairpins with 200 and 500 nt long stems from the gag coding region ofclade B HIV-III_(B) virus. The gagB200 and gagB500 pro-siRNAs morepotently suppressed HIV-III_(B), than the previous gag siRNA (FIG. 2D).More importantly, unlike the gag siRNA, both gag pro-siRNAs knocked downgag mRNA and inhibited viral spread in vitro for UG29 (clade A) and IN22(clade C) viruses, although they worked slightly less effectively thanagainst III_(B) virus. These data indicate that pro-siRNAs could beparticularly beneficial for targeting heterogeneous and rapidly evolvingviral genes.

Because mammalian cells are sensitive to bacterial endotoxin, whichstimulates off-target innate immunity via Toll-like receptor signaling,whether purified pro-siRNAs are contaminated with endotoxin wasassessed. Although SDS-eluted pro-siRNAs contained significant amountsof endotoxin, assayed by Limulus amoebocyte lysate (LAL) assay, HPLCpurified pro-siRNAs, even at concentrations as high as 320 nM, werebelow the limit of detection (0.25 EU/ml) (Table 1). Endotoxincontamination was tested for by assaying for induction of mRNAexpression of the proinflammatory cytokines TNFA, IL6, IL8 and IL12,measured 4 hr later by qRT-PCR in highly endotoxin-sensitivemonocyte-derived human macrophages (FIG. 10A). Incubation withHPLC-purified vif pro-siRNAs (320 nM) did not trigger cytokine geneexpression. Thus purified pro-siRNAs did not contain significant amountsof immunostimulatory endotoxin. Next MDMs were transfected with a fewsiRNAs and pro-siRNAs (at 20 nM) to test immune response mediated byendogenous immune sensors (FIG. 10B). siRNA and pro-siRNA against LMNAefficiently down regulated LMNA mRNA, indicating these siRNAs weresuccessfully transfected into MDMs. Comparing to siRNAs, pro-siRNAs didnot trigger excessive activation of immune genes.

To ascertain the sequence composition of pro-siRNAs, pro-siRNAs werecloned and deep sequenced using a cloning method established foreukaryotic siRNAs (sequencing reads and alignment summary in Table 2).Most reads were concentrated between 20 and 22 nt (FIGS. 4A and 11). Themajority of reads (on average ˜75%) aligned to the target sequence,plasmid backbone or the E. coli genome. The vast majority of alignedsequences (82-99%) originated from the target sequence (FIG. 4B);consistent with the efficient gene knockdown they induced. Reads weregenerated from the entire target sequence, but were also concentrated atspecific sites (‘hot spots’) (FIGS. 4C, 11, and 12A-E). There was somesequence strand bias for most of the hot spots (FIG. 12A). Because thedata (FIGS. 2C and 2F) strongly suggested that pro-siRNAs are doublestranded, it was possible that strand bias may have been due todifferences in ligation efficiency during cloning, a well-knownproblem²⁵, rather than the presence of many single-stranded RNAs. Toevaluate this further, forward and reverse DNA oligonucleotide probes(26-27 nt) were designed for three EGFPFL pro-siRNA hot spots andperformed solution hybridization and native gel electrophoresis (Table 3and FIG. 12B). The relative intensity of hybridized bands wasapproximately equal for sense and antisense probes for each hot spot andwere generally correlated with the number of reads from each hot spot(FIGS. 12C-12E). Thus, pro-siRNAs are mostly dsRNAs and the strand biasin the deep sequencing data likely reflects ligation bias duringcloning.

To further investigate the hot spot pattern, siRNA profiles of twoindependent preparations of EGFPFL pro-siRNAs cloned using differentsets of adapters were compared. The potency, size profile and sequencecontent of two EGFPFL pro-siRNAs were similar, but not identical. Themost abundant hot spots were consistent in the 2 samples, but the strandbias changed with the adapters, consistent with cloning bias (FIG.13A-D). Without wishing to be bound by theory, hot spots could be due tointrinsic sequence preferences for RNase III cleavage or differences instability or p19 binding after cleavage. To determine whether ‘hotspots’ are determined by sequence differences at or close to the hotspot, hairpins of equal sizes were constructed from the 5′ and 3′ endsof the full length EGFP sequence. The pro-siRNAs generated from the twohalves yielded mostly identical hot spots to the corresponding hot spotsin EGFPFL pro-siRNAs (FIG. 13E). Thus hot spots seem to be determined bylocal sequence differences. However a basic bioinformatic analysis ofsequence motif or preferred base for the hot spots was inconclusive(data not shown). E. coli RNase III might process dsRNA into siRNA-sizedsmall RNAs in vivo through a mechanism that differs from Dicer²⁷, whosecleavage of a long dsRNA results in phased and evenly distributedsequences along a target gene.

Because pro-siRNAs contained non-targeting sequences derived from theplasmid or E. coli genome, possible off-targeting effects²⁶ wereinvestigated. To evaluate off-targeting, RNA expression profiles werecompared by RNA deep sequencing of HeLa-d1EGFP cells transfected with 4nM of negative control or EGFP siRNA or EGFPFL or EGFP100 pro-siRNAs(sequencing reads and alignment summary in Table 2). Tophat andCufflinks were used to analyze the data and plotted volcano plots of allannotated transcripts (fold change versus p value, FIG. 4D). Comparingto EGFP siRNA, EGFP100 pro-siRNA had higher number of significantlychanged genes while EGFPFL pro-siRNA had less (FIGS. 4F and 14A). EGFPFLpro-siRNA also produced the least changes in long non-coding RNAs, agroup of newly discovered gene regulators (FIGS. 14B-14C). EGFP100pro-siRNAs, made from a shorter hairpin (100 bp), contained higherproportion of plasmid and genomic sequences compared to other pro-siRNAsmade from longer hairpins (200 to 720 bp, FIG. 4B), which is likely thecause of higher off-target effect. These data indicate a plasmidcontaining longer sequences of the target gene could have feweroff-target effects. Gene expression profiles of cells transfected withLMNA siRNAs and pro-siRNAs were also compared by microarray. Consistentwith the EGFP data, LMNA pro-siRNAs, made from a longer hairpin (523bp), produced fewer number of significantly changed genes comparing toLMNA siRNA (FIGS. 4E, 4F, and 14D). The RNA profiling data also showedthe target gene was always the most down regulated gene and pro-siRNAsconsistently produced better knockdown than siRNA. Thus pro-siRNAs couldbe engineered to offer better knockdown and lower off-target effectscompared to synthetic siRNAs. The significantly changed genes in each ofthese experiments were not enriched for innate immune genes³⁰,confirming that the pro-siRNAs did not stimulate an innate immuneresponse. Thus pro-siRNAs offer highly specific knockdown that is atleast as good as synthetic siRNAs without the need to test multiplesequences.

It is demonstrated herein that bacteria can be genetically engineered toproduce siRNAs that are highly effective and not toxic to mammaliancells. Specifically, it demonstrated herein is efficient knockdown ofone exogenous gene (EGFP), two viral genes (vif and gag) and 3 hostgenes (PLK1, TP53, LMNA). Without wishing to be bound by theory, becausepro-siRNAs are natural products of RNase III, they likely have favorableends (e. g., 5′-phosphate, 3′-hydroxyl and 3′ overhangs) for efficientloading by Ago into the RISC and do not activate cytosolic innate immuneRNA sensors. An alternative strategy of producing pro-siRNAs that usestwo plasmids—one to express p19 and the other to transcribe both senseand antisense strands of a target sequence—facilitates cloning and canalso be used to produce efficient gene silencing (FIGS. 15A-15C).

Without much optimization an average yield of ˜4 nmol (˜42 μg) pro-siRNAper liter of E. coli culture was achieved. It is contemplated that theengineered plasmid or E. coli genome could potentially be furtheroptimized to maximize yield and improve effectiveness. By way ofnon-limiting example, the yield of EGFPFL pro-siRNA could be doubled byoverexpressing E. coli RNase III (FIG. 16).

Generating pro-siRNAs for research purposes might be more cost effectivethan purchasing and testing multiple individual chemically synthesizedsiRNAs. pro-siRNAs, containing multiple sequences, might offer feweroff-target effects than individual siRNAs and could be harder for thetarget gene to escape silencing by mutation. On the other hand, chemicalsynthesis provides the opportunity for chemical modifications toincrease potency, enhance stability and reduce off-target effects orcouple fluorophores or targeting moieties. Such modifications might alsobe possible for pro-siRNAs, either by adding modified ribonucleotides tobacterial cultures during IPTG induction or by performing the samecoupling reactions with purified pro-siRNAs as are used to modifysiRNAs, respectively.

RNase III-deficient E. coli expressing dsRNAs can be fed to C. elegans¹⁸ and bacteria-derived dsRNAs can be applied to plants to inducespecific gene knockdown²⁸. However, gene silencing requires host Dicerand, unlike for mammalian cells, is enhanced in these organisms byRNA-dependent RNA polymerases that can amplify small amounts of RNA.More recently, genetically engineered E. coli, designed to express aninvasin to induce bacterial uptake and listeriolysin, to allow bacterialRNAs to escape from phagolysosomes, delivered dsRNAs into the cytoplasmof human cells through “trans-kingdom RNAi” technology²⁹.

pro-siRNAs, described here, could become a valuable cost effectiveaddition to existing RNAi techniques for both research and therapeutics.The method described herein for producing pro-siRNAs can easily beadopted and scaled-up in an industrial setting. It is contemplated thatmammalian cDNA libraries could be used to generate pro-siRNA libraries,e.g. for siRNA screening pro-siRNAs, generated from longer hairpinscontaining multiple sequences, might offer fewer off-target effects thanindividual siRNAs and in the cases of virus infection or cancer might beharder for the target gene to escape from by mutation. On the otherhand, chemical synthesis provides the opportunity for chemicalmodifications to increase potency, enhance stability and reduceoff-target effects or to couple fluorophores or targeting moieties. Suchmodifications can be applied to pro-siRNAs, e.g. either by addingmodified ribonucleotides to bacterial cultures during IPTG induction orby performing the same coupling reactions with purified pro-siRNAs asare used to modify siRNAs, respectively.

Methods

Bacterial strains and culture conditions. All E. coli strains used inthis study are listed in Table 4. E. coli strain DH5α was used forcloning and for initial characterization of the siRNA-like RNA species.For recombinant protein expression and pro-siRNA production, T7 ExpressIq (NEB), a BL21-derived E. coli strain was used. Two mutants of RNaseIII, rnc-14::DTn10 (Tet^(R)) and Drnc-38 (Kan^(R)) were utilized. Thesewere moved by P1 transduction from parent strains HT115(DE3)¹⁸ andSK7622¹⁹ into E. coli strain MG1655 ΔlacZYA (also referred as MG1655Δlac). All E. coli strains were cultured in LB broth, Lennox (BD) at 37°C. with shaking at 250 rpm and antibiotics when required were used atthe following concentrations; carbenicillin (100 μg/ml), kanamycin (50μg/ml), spectinomycin (50 μg/ml), tetracycline (12.5 μg/ml).

Listeria monocytogenes strain 10403S was cultured in brain-heartinfusion medium (BD Biosciences) at 30° C. Transformation of bacterialcells was performed as previously described³².

Genes and plasmids. The p19 gene used in this study was cloned fromTomato bushy stunt virus. All plasmids are listed in Table 5. To producep19 in E. coli, pcDNA3.1+(Invitrogen) was used to express the p19protein with a C-terminal FLAG tag (pcDNA3.1-p19-FLAG) or an N-terminalHis tag (pcDNA3.1-His-p19). Plasmid pcDNA3.1-TREX1-FLAG encodes aC-terminal FLAG-tagged TREX1 protein. To express p19 in L.monocytogenes, pLIV-1-His-p19 plasmid was used, which encodes p19 withan N-terminal His tag cloned in pLIV-1 plasmid (gift of Darren Higgins,Harvard Medical School). E. coli RNase III with an N-terminal FLAG wascloned in pcDNA3.1+ and pCDF-1b (Novagen) plasmids.

Two strategies were used for pro-siRNA production in E. coli. In oneapproach p19-His was fused to GST in pGEX-4T-1 (to express GST-p19-Hisfusion protein). On the same plasmid we cloned a hairpin RNA expressingcassette consisting of inverted repeat separated by a 32 bp linkerdownstream of a T7 promoter. A scheme of the resulting plasmid,pGEX-4T-1-p19-T7, is showed in FIG. 13A-13D. The hairpin RNA sequenceswere: EGFPFL, the entire 720-bp EGFP coding sequence (from pEGFP-N1,Clontech); EGFP100, a 100 bp from nt 219 to 318; EGFP Hotspot-1 360 bpfrom nt 1 to 360; EGFP Hotspot-2 360 bp from nt 361 to 720; LMNA(NM_005572.3), 523 bp from nt 267 to 789; TP53 (NM_000546.5), 301 bpfrom nt 376 to 676; PLK1 (NM_005030.3), 299 bp from nt 92 to 390; vif(K03455), the entire 579-bp; gag (K03455), gagB200: 200 bp from nt 1183to 1382, gagB500: 500 bp from nt 1004 to 1503. (Genbank entries listed;numbers refer to position with respect to the translation start site).

In another approach two compatible plasmids were used for pro-siRNAproduction. The GST-p19-His protein was cloned under the control of theT7 promoter in pRSF-1b (Novagen) or pCDF-1b to generate pRSF-GST-p19-Hisand pCDF-GST-p19-His. The second plasmid is a L4440 plasmid encoding theentire EGFP coding sequence (L4440-EGFP).

All cloning was performed using PCR and standard techniques. All primers(with information for restriction enzyme sites) are listed in Table 6.

Cells. HeLa-d1EGFP, HCT116, HCT116 Dicer^(−/−), HeLa-CD4 TZM-bl,U87.CD4.CXCR4 and U87.CD4.CCR5 cells were cultured in DMEM medium(Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum(FBS). ACH2 cells (human leukemia T cell line CEM latently infected withHIV-1) were cultured in RPMI medium (Invitrogen) supplemented with 10%heat-inactivated fetal bovine serum (FBS). For assays using primarymonocyte-derived human macrophages (MDM), monocytes were isolated fromblood of a healthy donor by Ficoll-Paque Plus (GE Healthcare) densityseparation. Monocytes were plated on PRIMARIA plates (FALCON) in RPMImedium (Invitrogen) supplemented with 10% heat-inactivated human serumand adherent cells were cultured for 5 d to allow differentiation intomacrophages.

RNA isolation and qRT-PCR. Total RNA was isolated from 3 ml of E. colistationary phase culture with 1 ml Trizol reagent (Invitrogen) followingthe manufacturer's protocol. RNA from human cells was collected inTrizol and extracted according to the manufacturer's protocol. Total RNA(1 μg) was converted to cDNA using SuperScript III Reverse Transcriptase(Invitrogen). For qRT-PCR, 10 μl reaction, containing SsoFast EvaGreenmastermix (Bio-Rad), appropriate primers (Table 4), and template cDNAsmade from 10 ng RNA, was amplified on a Bio-Rad CFX 96 Thermal Cycler.All qRT-PCR data were normalized to the human GAPDH gene. qRT-PCRprimers for human genes (Table 6) were selected from PrimerBank(available on the world wide web at pga.mgh.harvard.edu/primerbank/).

siRNA isolation from total RNA using p19 magnetic beads. p19 magneticbeads were prepared at NEB as previously described¹⁵. To pull downsiRNAs, 50 μg of total RNA (isolated from human or E. coli cells) wasused following the manufacturer's protocol¹⁵.

His-tag purification of GST-p19-His and bound pro-siRNA. GST-p19-His waspurified as follows. A fresh single transformant of T7 Express Iqcontaining pGEX-4T-1-p19-T7 was used to inoculate 300 ml LB medium in a1.5 L flask. When the OD₆₀₀ reached 0.3-0.6, protein and pro-siRNAexpression were induced by adding 0.5 mM IPTG for 1 hr. Cells werecentrifuged and lysed in 10 ml lysis buffer (50 mM Phosphate buffer pH7.0, 300 mM NaCl, 10 mM imidazole, 1% Triton X-100, 1 mg/ml lysozyme) at4° C. for ˜30 min followed by sonication (Misonix S-4000) until thelysate was non-viscous. Following centrifugation the lysate wasincubated with rotation with 1 ml Ni-NTA resin (Thermo Scientific)overnight at 4° C. The resin was washed with lysis buffer 4 times, eachtime for 10 min at 4° C. with rotation. Bound GST-p19-His was eluted inlysis buffer containing 300 mM imidazole at room temperature.

To purify p19-bound pro-siRNA the procedure was as above until the finalelution step when 500 μl 0.5% SDS was added for 10 min at roomtemperature with rotation. This step was repeated and both SDS eluateswere combined and passed through a 0.22 μm centrifuge filter (Corning)before HPLC purification on a Bio WAX NP5 anion exchange column (AgilentTechnologies). The HPLC buffers were: Buffer A, 25 mM Tris-HCl, 2 mMEDTA; Buffer B, 25 mM Tris-HCl, 2 mM EDTA, 5 M NaCl. HPLC was initiatedwith a flow rate of 1 ml/min at 25° C. Elution was performed using alinear gradient of 0-10% Buffer B over 4 min, followed by 10% Buffer Bfor 6 min, and a second linear gradient of 10-25% Buffer B over 15 minat a reduced flow rate of 0.5 ml/min. pro-siRNA eluted in the secondgradient was collected by isopropanol precipitation.

Polyacrylamide gel electrophoresis (PAGE) of RNA. For denaturingelectrophoresis of RNA, mini-sized pre-cast 15% polyacrylamide TBE-Ureagels (Invitrogen) were used. RNA samples were heated to 95° C. for 5 minin Gel Loading Buffer II (Ambion) and then immediately placed on iceuntil gel loading. Electrophoresis was performed in a 70° C. water bath(to ensure complete denaturation of siRNA) and gels were stained withSYBR Gold (Invitrogen). For analysis of E. coli total RNA, 20 μg samplesof Trizol-isolated RNA were loaded. RNA size standards (miRNA marker,siRNA marker and Low Range ssRNA Ladder) were from NEB.

For native electrophoresis of RNA, mini-sized homemade 15%polyacrylamide TBE gels were used with the Bio-Rad Mini-PROTEAN TetraCell. RNA samples were prepared in Gel Loading Buffer II (Ambion)without heat denaturation and electrophoresis was performed at roomtemperature.

Nuclease sensitivity assay. The nucleases tested were: RNase A, RNaseT1, and Turbo DNase (all from Ambion), Xrn1, exonuclease T, andexonuclease I (all from NEB). For each assay, 200 ng of an unmodifiedsynthetic negative control siRNA (GenePharma) and vifpro-siRNA were usedand assays were incubated in a 20 μl reaction volume using standardamounts of enzymes at 37° C. for 1 hr. Treated RNAs were purified byphenol/chloroform extraction followed by isopropanol precipitation.

Test for endotoxin activity and immune activation in primary humanmonocyte-derived macrophages (MDM)/RNA samples diluted in ddH₂O to theindicated concentration were analyzed by the single vial Gel Clot LALassay (detection limit 0.25 EU/ml, Lonza) following the manufacturer'sprotocol. Lipopolysaccharide (LPS) from E. coli O111:B4 (Sigma-Aldrich)was used as a positive control.

To test for cytokine gene activation, MDM plated in 24 well plates(1×10⁵ cells/well) were incubated with medium containing RNA or LPS atthe indicated concentration for 4 hr before harvesting RNA. siRNAs andpro-siRNAs were also transfected to MDMs at 20 nM using Lipofectamine2000 (Invitrogen) and total RNA were harvest at 24 hrs aftertransfection.

5′ ³²P labeling of RNA. RNA samples were dephosphorylated by AntarcticPhosphatase (NEB) for 30 min at 37° C. in the presence of Murine RNaseInhibitor (NEB). The Antarctic Phosphatase was deactivated by incubationat 65° C. for 5 min and the RNA was end-labeled with γ-³²P ATP(PerkinElmer) and T4 Polynucleotide Kinase (NEB). Gels were exposedusing a phosphorimager screen and visualized using a FLA-9000 ImageScanner (Fujifilm).

Small RNA northern blot. Northern blot for small RNAs was performed aspreviously described³³. The EGFP specific sense probe was a³²P-UTP-internally labeled RNA prepared by in vitro transcription usingT7 RNA polymerase (NEB) and a PCR-generated DNA template of thefull-length EGFP gene that incorporated a T7 promoter.

siRNA transfection for testing RNA silencing efficiency. All siRNAtransfections were performed using Lipofectamine 2000 following themanufacturer's protocol. Briefly, cells were plated in 24 well plates(1×10⁵ per well) and the transfection complex (containing 1.0 mlLipofectamine 2000 and siRNAs) was added directly to the medium. RNA andprotein samples were isolated from cells 24 hr post-transfection. Forthe PLK1 cell killing experiment, cells were counted using a TC-10automatic cell counter (Bio-Rad). The following siRNAs were used:ON-TARGETplus Non-targeting siRNA #4 (D-001810-04-05, Dharmacon),siGENOME Lamin A/C Control siRNA (D-001050-01-20, Dharmacon), Set of 4:siGENOME LMNA siRNA (MQ-004978-01-0002, Dharmacon), ON-TARGETplusSMARTpool-Human PLK1 (L-003290-00-0005, Dharmacon), Set of 4 Upgrade:ON-TARGETplus PLK1 siRNA(LU-003290-00-0002, Dharmacon), Set of 4:siGENOME TP53 siRNA (MQ-003329-03-0002, Dharmacon), Negative controlsiRNA (NC siRNA, B01001, GenePharma), Positive control siRNA TP53(B03001, GenePharma), custom EGFP siRNA (sense, GGCUACGUCCAGGAGCGCACC(SEQ ID NO: 114); antisense, UGCGCUCCUGGACGUAGCCUU (SEQ ID NO: 115)),custom vif siRNA-1²³ (sense, GUUCAGAAGUACACAUCCCT (SEQ ID NO: 116);antisense, GGGAUGUGUACUUCUGAACTT (SEQ ID NO: 117)) and custom siRNA-2²⁴(sense, CAGAUGGCAGGUGAUGAUUGT (SEQ ID NO: 118); antisense,AAUCAGCACCUGCCAUCUGTT (SEQ ID NO: 119)), custom gag siRNA: (sense,GAUUGUACUGAGAGACAGGCU (SEQ ID NO: 120); antisense, CCUGUCUCUCAGUACAAUCUU(SEQ ID NO: 121)).

RISC Immunoprecipitation. Cells (3×10⁶) were transfected with 4 nM NCsiRNA or EGFPFL pro-siRNAs. After 24 hours cells were scraped from theplate in 2 ml lysis buffer (150 mM KCl, 25 mM Tris-HCl pH 7.5, 2 mMEDTA, 0.5 mM DTT, 1% NP-40 and Roche Complete Protease InhibitorCocktail). Cells were then mechanically disrupted for 1 min using amicro-MiniBeadbeater (BioSpec). The cell lysate was incubated at 4° C.with rotation for 1 hr to ensure complete lysis. IP was performed byadding anti-Ago (2A8) antibody (Millipore, MABE56) or mouse total IgG(Jackson Labs) at 1:100 dilution together with 30 μl protein G Dynabeads(Invitrogen) and samples were rotated at 4° C. overnight. After washing4 times in lysis buffer, precipitated RNAs were isolated using Trizolreagent from 90% of the reaction mix, while 10% was saved for immunoblotinput.

Western Immunoblot. Protein samples were prepared by heating cells to95° C. for 5 min in 1×SDS loading buffer before SDS-PAGE. Immunoblot wasperformed using SNAP i.d. Protein Detection System (Millipore) followingthe manufacturer's protocol. Antibodies and their dilutions were:anti-FLAG (M2) 1:1,000 (Sigma-Aldrich, F1804), anti-His tag 1:500(Covance, MMS-156P), anti-PLK1 1:100 (Santa Cruz, sc-17783),anti-LaminA/C 1:1,000 (Santa Cruz, sc-7292), anti-p53 (DO-1) 1:500,(Santa Cruz, sc-126), anti-beta-Tubulin 1:10,000 (Sigma-Aldrich, T5168),anti-Ago (2A8) 1:1,000 (Millipore, MABE56). Horseradish peroxidaseconjugated anti-mouse or anti-rabbit IgG secondary antibodies were usedat 1:5,000 dilution followed by incubating the membranes in SuperSignalWest Pico Chemiluminescent Substrate (Thermo Scientific).

Solution hybridization and native gel electrophoresis assay. DNAoligonucleotides purchased from IDT were PAGE purified. Purified DNAoligonucleotides (10 pmol) were end-labeled with γ-³²P ATP by T4Polynucleotide Kinase (NEB) and 2 pmol was then mixed with 5 ng ofpro-siRNAs in buffer containing 20 mM Tris-HCl pH 7.9, 100 mM NaCl and 2mM EDTA. Samples were heated to 80° C. for 10 min and allowed to cool toroom temperature. A fraction of the sample was separated on a native 15%polyacrylamide gel. The gel was directly exposed to a phosphorimagerscreen. Multi-gauge software (Fujifilm) was used for imagequantification.

siRNA library preparation, deep sequencing, and data analysis. siRNAswere cloned according to the Illumina small RNA sample preparation guidev1.5 with the following exceptions. Custom 5′ RNA ligation adapters weresynthesized with a 4 nt nucleotide barcode sequence (Table 7). Small RNAlibraries were pooled and sequenced on one sequencing lane of anIllumina GAII sequencer (Genome Technology Core, Whitehead Institute orNEB). Novocraft software (www.novocraft.com) was used for sequencealignment. Reference genome was E. coli K12 substr. MG1655. We wrotePerl software scripts for data analysis. Original data and softwarescripts are available upon request.

mRNA profiling by microarray and deep sequencing. siRNAs and pro-siRNAs(4 nM) were transfected into HeLa-d1EGFP cells and RNA was isolated 24hr post-transfection. Non-targeting siRNA #4 (Dharmacon) was used asnegative control siRNA. Data from biological duplicates were analyzed atthe Microarray Core, Dana Farber Cancer Institute for microarrayanalysis using GeneChip 1.0 ST (Affymetrix). Microarray data wasanalyzed using dChip software and p values of gene expression changeswere calculated using paired T-test method³⁴. Original data and analysisfiles are available upon request.

For RNA deep sequencing, Ribo-Zero rRNA Removal Kits (Epicentre) wasused to remove large ribosomal RNAs from total RNA following themanufacturer's protocol. rRNA-depleted RNA (from 500 ng total RNA) wasused to construct deep sequencing library using NEBNext Ultra RNALibrary Prep Kit for Illumina (NEB #E7530) according to themanufacturer's protocol. Illumina GAII was used for sequencing (NEB).Tophat and Cufflinks software suites were used to analyzed the RNA deepsequencing data from biological duplicates. Reference genome was Humangenome GRCh37/hg19 and annotations of lincRNA transcripts weredownloaded from UCSD genome browser. Original data and analysis filesare available upon request.

Flow cytometry. For EGFP, cells were removed from plates by trypsindigestion and re-suspended in FACS buffer, DPBS (Invitrogen) containing2% heat-inactivated FBS. Intracellular staining of p24 antigen wasperformed using an Intracellular Staining Kit (Invitrogen) according tothe manufacturer's protocol and fluorescein-labeled p24 antibody (1:200,Beckman Coulter, cat#KC57-FITC). Fluorescence was analyzed on aFACSCalibur (BD) using FlowJo software (Tree Star).

HIV infection and TZM-bl assay. HeLa-CD4 cells were transfected with 4nM siRNA and pro-siRNA in 24 well plates (1×10⁵ cells/well). Cells wereinfected 12 hr post-transfection with HIV_(IIIB) (˜400 ng/ml p24) andculture medium was changed 12 hr post-infection. For HIV_(UG29)U87.CD4.CXCR4 cells were used and for HIV_(IN22) U87.CD4.CCR5 cells wereused. Culture medium was collected for TZM-bl assay and RNA wasextracted for qRT-PCR 24˜36 hr post-infection. TZM-bl cells, plated in24 well plates (1×10⁵ cells/well) 12 hr before, were analyzed 24 laterby luciferase assay performed using a Luciferase Assay System kit(Promega) following the manufacturer's protocol.

RNase A digestion assay for E. coli total RNA. ˜2 ug of total E. coliRNA were incubated with 1.0 unit of RNase A for 15 min at 37° C. in1×DNase I reaction buffer (NBE) supplemented with 400 mM NaCl. Theresulting products were analyzed on a 0.8% agarose gel containing EtBr.

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TABLE 1 Gel clot Limulus amoebocyte lysate (LAL) endotoxin assays ofHPLC-purified pro-siRNA Gel clot LAL assay Sample (limit of detection0.25 EU/ml) H2O − LPS (4 ng/ml) + P19 RNA SDS eluate + (~100 nM) HPLCpro-siRNA 16 nM − HPLC pro-siRNA 64 nM − HPLC pro-siRNA 160 nM − HPLCpro-siRNA 320 nM −

TABLE 2 Sequencing reads and alignment summary of RNA deep sequencingdata Aligned Percentage Type Sample name Total reads reads aligned TotalRNA NC siRNA-1 21,954,641 19,032,496 86.7% Total RNA NC siRNA-226,914,681 22,462,181 83.5% Total RNA EGFP siRNA-1 25,659,586 21,237,24182.8% Total RNA EGFP siRNA-2 23,235,174 19,588,652 84.3% Total RNAEGFP100 27,110,365 23,381,006 86.2% pro-siRNA-1 Total RNA EGFP10022,690,638 19,433,997 85.6% pro-siRNA-2 Total RNA EGFPFL 27,914,51123,335,378 83.6% pro-siRNA-1 Total RNA EGFPFL 21,572,278 18,178,02984.3% pro-siRNA-2 small RNA EGFPFL/ 3,291,738 3,119,677 94.8% EGFPFL-1small RNA EGFP100 2,967,297 1,488,213 50.2% small RNA LMNA 1,659,8901,382,441 83.3% small RNA TP53 5,446,487 4,462,318 81.9% small RNA PLK12,938,903 2,309,515 78.6% small RNA vif 1,869,202 1,493,137 79.9% smallRNA gagB200 5,326,736 3,640,886 68.4% small RNA gagB500 7,168,8295,017,221 70.0% small RNA EGFPFL-2 5,507,507 4,075,642 74.0% small RNAEGFP Hotspot-1 6,483,321 5,425,661 83.7% small RNA EGFP Hotspot-26,485,138 4,019,427 62.0%

TABLE 3 EGFPFL pro-siRNAs for testing strand bias SEQ Number Sequence IDof Rank- Name (5′-3′) NO Direction Start Reads ing Si1 UAGUGGUUGUC 7Antisense 602  279598  2 GGGCAGCAGC Si2 UAUAGACGUUG 8 Antisense 4571305273  1 UGGCUGUUG Si3 UGGUCGAGCUG 9 Sense  47   55643 11 GACGGCGACG

TABLE 4 List of E. coli strains Name Genotype Source/reference DH5αfhuA2Δ(argF-lacZ)U169 phoA NEB (C2987) glnV44 Φ80 Δ(lacZ)M15 gyrA96recA1 relA1 endA1 thi-1 hsdR17 T7 Express Iq MiniFlacI^(q)(Cam^(R))/fhuA2 lacZ::T7 gene1 NEB (C3016) [lon] ompT gal sulA11R(mcr-73::miniTn10-- Tet^(S))2 [dcm] R(zgb-210::Tn10--Tet^(S)) endA1Δ(mcrC-mrr)114::IS10 HT115(DE3) W3110 rnc-14::ATnJO λDE3 Timmons et al.(2001), gift of Gary Ruvkun BL21(DE3) fhuA2 [lon] ompT gal (λ DE3) [dcm]ΔhsdS NEB (C2527) λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7gene1) i21 Δnin5 SK7622 thyA715 Δrnc-38::Kmr Babitzke et al. (1993),gift of Sidney Kushner MG1655 ΔlacZYA F-lambda-ilvG-rfb-50 rph-1 ΔlacZYAGift from S. Garrity MG1655 ΔlacZYA rnc14 F-lambda-ilvG-rfb-50 rph-1ΔlacZYA This study Δrnc14 MG1655 ΔlacZYA rnc38 F-lambda-ilvG-rfb-50rph-1 ΔlacZYA This study Δrnc38

TABLE 5 List of plasmids Name Purpose pcDNA3.1+ Empty control plasmidpcDNA3.1- For expression of TREX1-FLAG protein directed from TREX1-FLAGthe CMV promoter pcDNA3.1-P19- For expression of P19-FLAG from the CMVpromoter FLAG pcDNA3.1-His- For expression of His-P19 from the CMVpromoter P19 pcDNA3.1-P19- For expression of His-P19 3942 mutant fromthe CMV 3942-His promoter pcDNA3.1-P19- For expression of His-P19 7172mutant from the CMV 7172-His promoter pcDNA3.1-RNase For expression ofFLAG tagged E. coli RNase III III pRSF-GST-P19- For expression ofGST-P19-His from T7 promoter His pCDF-GST-P19- For expression ofGST-P19-His from T7 promoter His pCDF-RNase III For expression of FLAGtagged E. coli RNase III from T7 promoter L4440-EGFP For expression ofdouble stranded eGFP RNA from convergent T7 promoters pGEX-4T-1-P19- Forexpression of GST-P19-His from Tac promoter His pGEX-4T-1-P19- Forexpression of GST-P19-His from Tac promoter, T7 and hairpin RNA from theT7 promoter pGEX-4T-1-P19- For producing EGFPFL pro-siRNA T7-EGFPFLpGEX-4T-1-P19- For producing EGFP Hotspot1 pro-siRNA T7-EGFP- Hotspot1pGEX-4T-1-P19- For producing EGFP Hotspot2 pro-siRNA T7-EGFP- Hotspot2pGEX-4T-1-P19- For producing EGFP100 pro-siRNA T7-EGFP100 pGEX-4T-1-P19-For producing LMNA pro-siRNA T7-LMNA pGEX-4T-1-P19- For producing PLK1pro-siRNA T7-PLK1 pGEX-4T-1-P19- For producing TP53 pro-siRNA T7-TP53pGEX-4T-1-P19- For producing HIV-vif pro-siRNA T7-Vif pGEX-4T-1-P19- Forproducing gagB200 pro-siRNA T7-GagB200 pGEX-4T-1-P19- For producinggagB500 pro-siRNA T7-GagB500 pLIV-1 Empty control plasmid pLIV-1-His-P19For expression of His-P19 protein in L. monocytogenes

TABLE 6 List of DNA oligonucleotides SEQ Name Sequence (5′-3′) ID NOPurpose P19-F-NheI AATCGCTAGCATGGAACGAGCTAT  10 pcDNA3.1-P19- ACAAGGAFLAG P19-R-BamHI AATCGGATCCCTCGCTTTCTTTTTC  11 pcDNA3.1-P19- GAAGG FLAGP19NLS-F AATCGGATCCGATCCAAAAAAGAA  12 pcDNA3.1-P19-GAGAAAGGTAGATCCAAAAAAGAA FLAG GAGAAAGGTA P19NLS-RAATCCTCGAGTCACTTATCGTCGTC  13 pcDNA3.1-P19- ATCCTTGTAATCGCCTACCTTTCTCFLAG TTCTTTTT P19-F-His- AATCGCTAGCATGCACCACCACCA  14 pcDNA3.1-P19- NheICCACCACGCGGGCGAACGAGCTAT His ACAAGGA P19-R-BamHIAATCGGATCCTCACTCGCTTTCTTT  15 pcDNA3.1-P19- TTCGAAGG His P19W3942G-FCCGAGTGGCACTGAGGGCCGGCTA  16 pcDNA3.1-P19- CATAACGATGAGACGAATTC 3942-HisP19W3942G-R TAGCCGGCCCTCAGTGCCACTCGGA  17 pcDNA3.1-P19-CTTTCGTCAGGAAGTTTGA 3942-His P19KR7172A GTTGTATTTGCGGGCTATCTCAGAT  18pcDNA3.1-P19- G-F ACGACAGGACGGAAGCTTC 7172-His P19KR7172ATCTGAGATAGCCCGCAAATACAAC  19 pcDNA3.1-P19- G-R TTTCCCGAAACCCCAGCTTT7172-His P19F-XbaI AATATCTAGAATGGAACGAGCTAT  20 pLIV-1-P19- ACAAGGA HisP19R-His- AATCTCTAGATCAGTGGTGGTGGTG  21 pLIV-1-P19- XbaI GTGGTG HisP19-F-BamHI AATCGGATCCATGGAACGAGCTAT  22 pGEX-4T-1- ACAAGGA P19-HisP19His-R- AATCCTCGAGTCAGTGGTGGTGGTG  23 pGEX-4T-1- XhoIGTGGTGCTCGCTTTCTTTTTCGAAG P19-His G mc-FLAG- ACTTGCTAGCATGGATTACAAGGAT 24 pcDNA3.1-RNase NheI-F GACGACGATAAGAACCCCATCGTA III and pCDF-ATTAATCG RNase III mc-BamHI-R ATCGGGATCCTCATTCCAGCTCCAG  25RNase III and TTTTTTCAA pCDF-RNase III His-T7-SacIATCGAGCTCCCCTATAGTGAGTCGT  26 pGEX-4T-1- ATTAGATTCAGTGGTGGTGGTGGTGP19-T7 GT Linker3-F ATGAATTCGTCGACACTGCGGCCGC  27 pGEX-4T-1-TCTAGAGGGCCCGTTTAAACCCGCT P19-T7 Linker3-R ATCTCGAGAATGAGCTCGCTGATCA  28pGEX-4T-1- GCGGGTTTAAACGGGCCCTCTAGA P19-T7 G GST-F-NdeIATCCCATATGTCCCCTATACTAGGT  29 pRSF-GST-P19- TATTG His, pCDF-GST- P19-HisHis-R-XhoI AATCCTCGAGTCAGTGGTGGTGGTG  30 pRSF-GST-P19- GTGGTGHis, pCDF-GST- P19-His EGFP-F-SacI AATCGAGCTCCATGGTGAGCAAGG  31pGEX-4T-1- GCGAGGA P19-T7-EGFPFL EGFP-F-NotI AATCGCGGCCGCATGGTGAGCAAG 32 pGEX-4T-1- GGCGAGGA P19-T7-EGFPFL EGFP-R-SalIAATCGTCGACCTACTTGTACAGCTC  33 pGEX-4T-1- GTCCA P19-T7-EGFPFL EGFP-F-XhoIAATCCTCGAGCTACTTGTACAGCTC  34 pGEX-4T-1- GTCCA P19-T7-EGFPFL,EGFP northern blot probe EGFPHS1-F- ATCCGCGGCCGCATGGTGAGCAAG  35pGEX-4T-1-P19- NotI GGCGAGGAG T7-EGFP-Hotspot1 EGFPHS1-F-ATCGAGCTCATGGTGAGCAAGGGC  36 pGEX-4T-1-P19- SacI GAGGAG T7-EGFP-Hotspot1EGFPHS1-R- ATCGTCGACCAGGGTGTCGCCCTCG  37 pGEX-4T-1-P19- SalI AACTTT7-EGFP-Hotspot1 EGFPHS1-R- ATCCTCGAGCAGGGTGTCGCCCTCG  38 pGEX-4T-1-P19-XhoI AACTT T7-EGFP-Hotspot1 EGFPHS2-F- ATCCGCGGCCGCGTGAACCGCATC  39pGEX-4T-1-P19- NotI GAGCTGAAG T7-EGFP-Hotspot2 EGFPHS2-F-ATCGAGCTCGTGAACCGCATCGAG  40 pGEX-4T-1-P19- SacI CTGAAG T7-EGFP-Hotspot2EGFPHS2-R- ATCGTCGACCTACTTGTACAGCTCG  41 pGEX-4T-1-P19- SalI TCCATT7-EGFP-Hotspot2 EGFPHS2-R- ATCCTCGAGCTACTTGTACAGCTCG  42 pGEX-4T-1-P19-XhoI TCCAT T7-EGFP-Hotspot2 EGFP100-F- AATCGAGCTCCCGCTACCCCGACCA  43pGEX-4T-1-P19- SacI CATGAA T7-EGFP100 EGFP100-F-AATCCGCGGCCGCCCGCTACCCCG  44 pGEX-4T-1-P19- NotI ACCACATGAA T7-EGFP100EGFP100-R- AATCGTCGACGTTGCCGTCGTCCTT  45 pGEX-4T-1-P19- SalI GAAGAAT7-EGFP100 EGFP100-R- AATCCTCGAGGTTGCCGTCGTCCTT  46 pGEX-4T-1-P19- XhoIGAAGAA T7-EGFP100 TP53-R-SalI AATCGTCGACCAACCTCAGGCGGC  47pGEX-4T-1-P19- TCATAGG T7-TP53 TP53-RXhoI AATCCTCGAGCAACCTCAGGCGGC  48pGEX-4T-1-P19- TCATAGG T7-TP53 TP53-F-Not AATCGCGGCCGCTACTCCCCTGCCC  49pGEX-4T-1-P19- TCAACAAGATG T7-TP53 TP53-F-SacI AATCGAGCTCTACTCCCCTGCCCTC 50 pGEX-4T-1-P19- AACAAGATG T7-TP53 HIV-Vif-F- AATCGAGCTCGGAAAACAGATGGC 51 pGEX-4T-1-P19- SacI AGGTGATG T7-Vif HIV-Vif-F-AATCGCGGCCGCGGAAAACAGATG  52 pGEX-4T-1-P19- NotI GCAGGTGATG T7-VifHIV-Vif-R- AATCGTCGACCTAGTGTCCATTCAT  53 pGEX-4T-1-P19- SalI TGTGTGGT7-Vif HIV-Vif-R- AATCCTCGAGCTAGTGTCCATTCAT  54 pGEX-4T-1-P19- XhoITGTGTGG T7-Vif LaminAC-F- AATCGAGCTCCAAGACCCTTGACTC  55 pGEX-4T-1-P19-SacI AGTAGCC T7-LMNA LaminAC-F- AATCGCGGCCGCCAAGACCCTTGA  56pGEX-4T-1-P19- NotI CTCAGTAGCC T7-LMNA LaminAC-R-AATCGTCGACCAGCTCCTTCTTATA  57 pGEX-4T-1-P19- SalI CTGCTCCA T7-LMNALaminAC-R- AATCCTCGAGCAGCTCCTTCTTATA  58 pGEX-4T-1-P19- XhoI CTGCTCCAT7-LMNA PLK1-F- AATCGCGGCCGCTCTCTGCTGCTCA  59 pGEX-4T-1-P19- NotIAGCCGCAC T7-PLK1 PLK1-F- AATCGAGCTCTCTCTGCTGCTCAAG  60 pGEX-4T-1-P19-SacI CCGCAC T7-PLK1 PLK1-R- AATCGTCGACAAGTCTCAAAAGGT  61 pGEX-4T-1-P19-SalI GGTTTGCC T7-PLK1 PLK1-R- AATCCTCGAGAAGTCTCAAAAGGT  62pGEX-4T-1-P19- XhoI GGTTTGCC T7-PLK1 Gag200- ATCCGCGGCCGCTGTGGCAAAGAA 63 pGEX-4T-1-P19- FNotI GGGCACACAG T7-GagB200 Gag200-ATCGAGCTCTGTGGCAAAGAAGGG  64 pGEX-4T-1-P19- FSacI CACACAG T7-GagB200Gag200- ATCGTCGACTCTTCTGGTGGGGCTG  65 pGEX-4T-1-P19- RSalI TTGGCTT7-GagB200 Gag200- ATCCTCGAGTCTTCTGGTGGGGCTG  66 pGEX-4T-1-P19- RXhoITTGGCT T7-GagB200 Gag500- ATCCGCGGCCGCAAGCATTGGGAC  67 pGEX-4T-1-P19-FNotI CAGCGGCTAC T7-GagB500 Gag500- ATCGAGCTCAAGCATTGGGACCAG  68pGEX-4T-1-P19- FSacI CGGCTAC T7-GagB500 Gag500-ATCGTCGACTTATTGTGACGAGGGG  69 pGEX-4T-1-P19- RSalI TCGTTG T7-GagB500Gag500- ATCCTCGAGTTATTGTGACGAGGGG  70 pGEX-4T-1-P19- RXhoI TCGTTGT7-GagB500 SiSEQ1 CAAGCAGAAGACGGCATACGA  71 Deep sequencing library PCRSiSEQ2 AATGATACGGCGACCACCGACAGG  72 Deep sequencing TTCAGAGTTCTACAGTCCGAlibrary PCR GAPDH For CTGGGCTACACTGAGCACC  73 GAPDH RevAAGTGGTCGTTGAGGGCAATG 126 IL12 For CACTCCCAAAACCTGCTGCTGAG  74 qRT-PCRIL12 Rev TCTCTTCAGAAGTGCAAGGGTA  75 qRT-PCR IL6 ForGATGAGTACAAAAGTCCTGATCCA  76 qRT-PCR IL6 Rev CTGCAGCCACTGGTTCTGT  77qRT-PCR IL8 For AGACAGCAGAGCACACAAGC  78 qRT-PCR IL8 RevATGGTTCCTTCCGGTGGT  79 qRT-PCR TNFA For CAGCCTCTTCTCCTTCCTGAT  80qRT-PCR TNFA Rev GCCAGAGGGCTGATTAGAGA  81 qRT-PCR Vif ForAGGGAAAGCTAGGGGATGGTTTT  82 qRT-PCR Vif Rev CCCAAATGCCAGTCTCTTTCTCC  83qRT-PCR IN22-Vif For AAAGAGAGCTAATGGATGGTTTT  84 qRT-PCR IN22-Vif RevCCCAAATGCCAATCTCTTTCCCC  85 qRT-PCR UG29-Vif For AAAGAAAGCTACTGGTTGGTGTT 86 qRT-PCR UG29-vif Rev CCCAAGTGCCAGTCTTTTTCTCC  87 qRT-PCR GagABC ForCCTAGGAAAAAGGGCTGTTGGA  88 qRT-PCR GagABC Rev AGGAAGGCCAGATCTTCCCTAAA 89 qRT-PCR IFIT1For GCCACAAAAAATCACAAGCCA  90 qRT-PCR IFIT1RevCCATTGTCTGGATTTAAGCGG  91 qRT-PCR LMNA For AGCAGCGTGAGTTTGAGAGC  92qRT-PCR LMNA Rev CCAGCTTGGCAGAATAAGTCTT  93 qRT-PCR PLK1 ForCGAGGACAACGACTTCGTGTT  94 qRT-PCR PLK1 Rev ACAATTTGCCGTAGGTAGTATCG  95qRT-PCR TP53 For ACAGCTTTGAGGTGCGTGTTT  96 qRT-PCR TP53 RevCCCTTTCTTGCGGAGATTCTCT  97 qRT-PCR eGFP For ACGTAAACGGCCACAAGTTC  98qRT-PCR eGFP Rev AAGTCGTGCTGCTTCATGTG  99 qRT-PCR EGFP-T7-FACTAATACGACTCACTATAGGGAT 100 EGFP northern GGTGAGCAAGGGCGAGGA blot probeEGFPFL-si1-F CGTGCTGCTGCCCGACAACCACTAC 101 Solution CT hybridizationEGFPFL-si1-R GAGGTAGTGGTTGTCGGGCAGCAG 102 Solution CACG hybridizationEGFPFL-si2-F CTACAACAGCCACAACGTCTATATC 103 Solution A hybridizationEGFPFL-si2-R TGATATAGACGTTGTGGCTGTTGTA 104 Solution G hybridizationEGFPFL-si3-F CCTGGTCGAGCTGGACGGCGACGT 105 Solution AA hybridizationEGFPFL-si3-R TTACGTCGCCGTCCAGCTCGACCAG 106 Solution G hybridizationACH-5 TATGAGGAACAGATTTTCTCACATG 107 Control oligo for G solutionhybridization

TABLE 7 RNA adapters for small RNA deep sequencing libraries SEQ NameSequence (5′-3′) ID NO Purpose 5ADPT-2 GUUCAGAGUUCUACA 108 5′adapter for GUCCGACGAUCGCUU EGFPFL 5ADPT-3 GUUCAGAGUUCUACA 109 5′adapter for GUCCGACGAUCGAGU EFGP100 5ADPT-5 GUUCAGAGUUCUACA 110 5′adapter for GUCCGACGAUCCGUU PLK1 5ADPT-6 GUUCAGAGUUCUACA 111 5′adapter for GUCCGACGAUCCCGU LMNA 5ADPT-7 GUUCAGAGUUCUACA 112 5′adapter for GUCCGACGAUCCACU HIV-Vif 3ADPT UCGUAUGCCGUCUUC 113 3′adapter for UGCUUGUidT all libraries

What is claimed herein is:
 1. A bacterial cell comprising asiRNA-binding polypeptide and a dsRNA comprising a nucleic acid sequencesubstantially complementary to a target RNA.
 2. The bacterial cell ofclaim 1, wherein the siRNA-binding polypeptide comprises a purificationtag.
 3. The bacterial cell of claim 1, wherein the siRNA-bindingpolypeptide is encoded by a nucleic acid.
 4. The bacterial cell of claim1, wherein the siRNA-binding polypeptide is selected from the groupconsisting of: p19 polypeptide; tombusvirus p19 polypeptide; B2polypeptide; HC-Pro polypeptide; p38 polypeptide; p122 polypeptide; p130polypeptide; p21 polypeptide; p1b polypeptide; and NS3 polypeptide. 5.The bacterial cell of claim 1, wherein the dsRNA is greater than 21nucleotides in length.
 6. The bacterial cell of claim 1, wherein thedsRNA is a hairpin RNA.
 7. The bacterial cell of claim 1, wherein thebacterial cell expresses an RNase III polypeptide.
 8. The bacterial cellof claim 1, wherein the bacterial cell expresses an RNase IIIpolypeptide encoded by an exogenous nucleic acid sequence.
 9. Thebacterial cell of claim 1, wherein the bacterial cell is an Escherichiacoli cell.
 10. The bacterial cell of claim 1, wherein at least one ofthe siRNA-binding polypeptide and the dsRNA are constitutivelyexpressed.
 11. The bacterial cell of claim 1, wherein at least one ofthe siRNA-binding polypeptide and the dsRNA are inducibly expressed. 12.The bacterial cell of claim 1, wherein the DNA encoding at least one ofthe siRNA-binding polypeptide or the dsRNA is part of a plasmid.
 13. Thebacterial cell of claim 1, wherein the dsRNA comprises nucleic acidsequences substantially complementary to a multiplicity of target RNAs.14. A method of producing one or more siRNA species which can inhibitthe expression of a target RNA, the method comprising: culturing abacterial cell of claim 1 under conditions suitable for the productionof siRNAs.
 15. The method of claim 14, further comprising a second stepof isolating the siRNA-binding polypeptide and eluting the siRNAs boundto the siRNA-binding polypeptide.
 16. The method of claim 15, furthercomprising purifying the siRNAs eluted from the siRNA-bindingpolypeptide by HPLC.
 17. The method of claim 14, further comprisingcontacting the cell with one or more modified nucleotides before orduring the culturing step.
 18. A pharmaceutical composition comprising asiRNA isolated from a bacterial cell of claim
 1. 19. The composition ofclaim 18, further comprising a population of siRNA species.
 20. A vectorcomprising; a nucleic acid encoding a siRNA-binding polypeptide; and adsRNA cloning site or a dsRNA comprising a nucleic acid sequencesubstantially complementary to a target RNA.